Treatment of hyperproliferative disorders with diarylhydantoin compounds

ABSTRACT

The present invention relates to diarylhydantoin compounds, including diarylthiohydantoins, and methods for synthesizing them and using them in the treatment of hormone refractory prostate cancer.

The present invention relates to diarylhydantoin compounds includingdiarylthiohydantoins, and methods for synthesizing them and using themin the treatment of hormone refractory prostate cancer. This applicationclaims priority from U.S. provisional applications bearing Ser. Nos.60/756,552, 60/750,351, and 60/680,835, the specifications of which arehereby incorporated by reference.

BACKGROUND OF THE INVENTION

Prostate cancer is the most common incidence of cancer and the secondleading cause of cancer death in Western men. When the cancer isconfined locally, the disease can be cured by surgery or radiation.However, 30% of such cancer relapses with distant metastatic disease andothers have advanced disease at diagnoses. Advanced disease is treatedby castration and/or administration of antiandrogens, the so-calledandrogen deprivation therapy. Castration lowers the circulating levelsof androgens and reduces the activity of androgen receptor (AR).Administration of antiandrogens blocks AR function by competing awayandrogen binding, therefore, reducing the AR activity. Althoughinitially effective, these treatments quickly fail and the cancerbecomes hormone refractory.

Recently, overexpression of AR has been identified and validated as acause of hormone refractory prostate cancer. See Chen, C. D., Welsbie,D. S., Tran, C., Baek, S. H., Chen, R., Vessella, R., Rosenfeld, M. G.,and Sawyers, Molecular determinants of resistance to antiandrogentherapy, Nat. Med., 10: 33-39, 2004, which is hereby incorporated byreference. Overexpression of AR is sufficient to cause progression fromhormone sensitive to hormone refractory prostate cancer, suggesting thatbetter AR inhibitors than the current drugs can slow the progression ofprostate cancer. It was demonstrated that AR and its ligand binding arenecessary for growth of hormone refractory prostate cancer, indicatingthat AR is still a target for this disease. It was also demonstratedthat overexpression of AR converts anti-androgens from antagonists toagonists in hormone refractory prostate cancer (an AR antagonistinhibits AR activity and an AR agonist stimulates AR activity). Datafrom this work explains why castration and anti-androgens fail toprevent prostate cancer progression and reveals unrecognized propertiesof hormone refractory prostate cancer.

Bicalutamide (brand name: Casodex) is the most commonly usedanti-androgen. While it has an inhibitory effect on AR in hormonesensitive prostate cancer, it fails to suppress AR when cancer becomeshormone refractory. Two weaknesses of current antiandrogens are blamedfor the failure to prevent prostate cancer progression from the hormonesensitive stage to the hormone refractory disease and to effectivelytreat hormone refractory prostate cancer. One is their weak antagonisticactivities and the other is their strong agonistic activities when AR isoverexpressed in hormone refractory prostate cancer. Therefore, betterAR inhibitors with more potent antagonistic activities and minimalagonistic activities are needed to delay disease progression and totreat the fatal hormone refractory prostate cancer.

Nonsteroidal anti-androgens, such as bicalutamide, have been preferredover steroidal compounds for prostate cancer because they are moreselective and have fewer side effects. This class of compounds has beendescribed in many patents such as U.S. Pat. No. 4,097,578, U.S. Pat. No.5,411,981, U.S. Pat. No. 5,705,654, PCT International Applications WO97/00071 and WO 00/17163, and U.S. Published Patent Application Number2004/0009969, all of which are hereby incorporated by reference.

U.S. Pat. No. 5,434,176 includes broad claims which encompass a verylarge number of compounds, but synthetic routes are only presented for asmall fraction of these compounds and pharmacological data are onlypresented for two of them, and one skilled in the art could not readilyenvision other specific compounds.

Because the mechanism of hormone refractory prostate cancer was notknown, there was no biological system to test these compounds describedin these patents for their effect on hormone refractory prostate cancer.Particularly, the ability of AR overexpression in hormone refractoryprostate cancer to switch inhibitors from antagonists to agonists wasnot recognized. Some new properties of hormone refractory prostatecancer are reported in PCT applications US04/42221 and US05/05529, whichare hereby incorporated by reference. PCT International ApplicationUS05/05529 presented a methodology for identifying androgen receptorantagonist and agonist characteristics of compounds. However, for eachcompound produced, the time consuming process of determining theantagonist and agonist characteristics of a compound must be determined.That is, there is no method to accurately predict characteristicsrelevant to treating prostate cancer from the chemical structure of acompound alone.

There is a need for new thiohydantoin compounds having desirablepharmacological properties, and synthetic pathways for preparing them.Because activities are sensitive to small structural changes, onecompound may be effective in treating prostate cancer, whereas a secondcompound may be ineffective, even if it differs from the first compoundonly slightly, say by the replacement of a single substituent.

Identification of compounds which have high potency to antagonize theandrogen activity, and which have minimal agonistic activity shouldovercome hormone refractory prostate cancer (HRPC) and avoid or slowdown the progression of hormone sensitive prostate cancer (HSPC).Therefore, there is a need in the art for the identification ofselective modulators of the androgen receptor, such as modulators whichare non-steroidal, non-toxic, and tissue selective.

SUMMARY OF THE INVENTION

The invention provides a series of compounds having strong antagonisticactivities with minimal agonistic activities against AR. These compoundsinhibit the growth of hormone refractory prostate cancer.

The invention includes a compound having the formula

wherein X is selected from the group consisting of trifluoromethyl andiodo, wherein W is selected from the group consisting of O and NR5,wherein R5 is selected from the group consisting of H, methyl, and

wherein D is S or O and E is N or O and G is alkyl, aryl, substitutedalkyl or substituted aryl; or D is S or O and E-G together are C1-C4lower alkyl,

wherein R1 and R2 together comprise eight or fewer carbon atoms and areselected from the group consisting of alkyl, substituted alkyl includinghaloalkyl, and, together with the carbon to which they are linked, acycloalkyl or substituted cycloalkyl group,

wherein R3 is selected from the group consisting of hydrogen, halogen,methyl, C1-C4 alkoxy, formyl, haloacetoxy, trifluoromethyl, cyano,nitro, hydroxyl, phenyl, amino, methylcarbamoyl, methoxycarbonyl,acetamido, methanesulfonamino, methanesulfonyl,4-methanesulfonyl-1-piperazinyl, piperazinyl, and C1-C6 alkyl or alkenyloptionally substituted with hydroxyl, methoxycarbonyl, cyano, amino,amido, nitro, carbamoyl, or substituted carbamoyl includingmethylcarbamoyl, dimethylcarbamoyl, and hydroxyethylcarbamoyl,

wherein R4 is selected from the group consisting of hydrogen, halogen,alkyl, and haloalkyl, and

wherein R3 is not methylaminomethyl or dimethylaminomethyl.

R5 may be

The compound may have the formula

wherein R3 is selected from the group consisting of hydroxy,methylcarbamoyl, methylcarbamoylpropyl, methylcarbamoylethyl,methylcarbamoylmethyl, methylsulfonecarbamoylpropyl, methylaminomethyl,dimethylaminomethyl, methylsulfonyloxymethyl, carbamoylmethyl,carbamoylethyl, carboxymethyl, methoxycarbonylmethyl, methanesulfonyl,4-cyano-3-trifluoromethylphenylcarbamoylpropyl, carboxypropyl,4-methanesulfonyl-1-piperazinyl, piperazinyl, methoxycarbonyl,3-cyano-4-trifluoromethylphenylcarbamoyl, hydroxyethylcarbamoylethyl,and hydroxyethoxycarbonylethyl, and

wherein R10 and R11 are both H or, respectively, F and H, or H and F. Incertain embodiments, R10 and R11 may both be H or, respectively, F andH. R3 may be methylcarbamoyl.

In some embodiments, R1 and R2 are independently methyl or, togetherwith the carbon to which they are linked, a cycloalkyl group of 4 to 5carbon atoms, and R3 is selected from the group consisting of carbamoyl,alkylcarbamoyl, carbamoylalkyl, and alkylcarbamoylalkyl, and R4 is H orF or R4 is 3-fluoro.

In other embodiments, R1 and R2 are independently methyl or, togetherwith the carbon to which they are linked, a cycloalkyl group of 4 to 5carbon atoms, R3 is selected from the group consisting of cyano,hydroxy, methylcarbamoyl, methylcarbamoyl-substituted alkyl,methylsulfonecarbamoyl-substituted alkyl, methylaminomethyl,dimethylaminomethyl, methylsulfonyloxymethyl, methoxycarbonyl,acetamido, methanesulfonamido, carbamoyl-substituted alkyl,carboxymethyl, methoxycarbonylmethyl, methanesulfonyl,4-cyano-3-trifluoromethylphenylcarbamoyl-substituted alkyl,carboxy-substituted alkyl, dimethylethoxy)carbonyl)-1-piperazinyl,4-methanesulfonyl-1-piperazinyl, piperazinyl,hydroxyethylcarbamoyl-substituted alkyl,hydroxyethoxycarbonyl-substituted alkyl, and3-cyano-4-trifluoromethylphenylcarbamoyl, and R4 is F.

Compounds of the invention may have the formula

wherein R3 is selected from the group consisting of methylcarbonyl,methoxycarbonyl, acetamido, and methanesulfonamido, and R4 is selectedfrom the group consisting of F and H.

Compounds of the invention may have the formula

wherein R4 is selected from the group consisting of F and H.

In embodiments of the invention, wherein R1 and R2 together with thecarbon to which they are linked are

Compounds of the invention may be those listed in Tier 1, Tier 2, Tier3, and/or Tier 4, below. Particular compounds of the invention include

The invention also provides a pharmaceutical composition comprising atherapeutically effective amount of a compound according to any of thepreceding compounds or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable carrier or diluent.

The invention encompasses a method for treating a hyperproliferativedisorder comprising administering such a pharmaceutical composition to asubject in need of such treatment, thereby treating thehyperproliferative disorder. The hyperproliferative disorder may behormone refractory prostate cancer. The dosage may be in the range offrom about 0.001 mg per kg body weight per day to about 100 mg per kgbody weight per day, about 0.01 mg per kg body weight per day to about100 mg per kg body weight per day, about 0.1 mg per kg body weight perday to about 10 mg per kg body weight per day, or about 1 mg per kg bodyweight per day.

The compound may be administered by intravenous injection, by injectioninto tissue, intraperitoneally, orally, or nasally. The composition mayhave a form selected from the group consisting of a solution,dispersion, suspension, powder, capsule, tablet, pill, time releasecapsule, time release tablet, and time release pill.

The administered compound may be selected from the group consisting ofRD162′, RD162″, RD 169, or RD170, or a pharmaceutically acceptable saltthereof. The administered compound may be RD162 or a pharmaceuticallyacceptable salt thereof.

The invention provides a method of synthesizing a diaryl compound offormula:

comprising mixing Compound I

with Compound II

in a first polar solvent to form a mixture, heating the mixture, addinga second polar solvent, the same as or different from the first polarsolvent, and an aqueous acid to the mixture, refluxing the mixture,cooling the mixture and combining with water, and separating the diarylcompound from the mixture, wherein R51 comprises an alkyl chain of from1 to 4 carbon atoms, R52 is selected from the group consisting of cyano,hydroxy, methylcarbamoyl, methylcarbamoyl-substituted alkyl,methylsulfonecarbamoyl-substituted alkyl, methylaminomethyl,dimethylaminomethyl, methylsulfonyloxymethyl, methoxycarbonyl,3-cyano-4-trifluoromethylphenylcarbamoyl, carbamoyl-substituted alkyl,carboxymethyl, methoxycarbonylmethyl, methanesulfonyl,4-cyano-3-trifluoromethylphenylcarbamoyl-substituted alkyl,carboxy-substituted alkyl, 4-methanesulfonyl-1-piperazinyl, piperazinyl,hydroxyethylcarbamoyl-substituted alkyl, andhydroxyethoxycarbonyl-substituted alkyl, and R53 is selected from thegroup consisting of F and H.

R51 may comprise an alkyl chain of from 1 to 2 carbon atoms, R52 may beselected from the group consisting of carbamoyl and methylcarbamoyl, andR53 may be F.

The invention provides methods of synthesizing a compound of formula:

comprising mixing 4-isothiocyanato-2-trifluoromethylbenzonitrile andN-methyl-4-(1-cyanocyclobutylamino)-2-fluorobenzamide indimethylformamide to form a first mixture, heating the first mixture toform a second mixture, adding alcohol and acid to the second mixture toform a third mixture, refluxing the third mixture to form a fourthmixture, cooling the fourth mixture, combining the fourth mixture withwater and extracting an organic layer; isolating the compound from theorganic layer.

Likewise, the invention provides a method of synthesizing RD162′comprising mixing N-Methyl-2-fluoro-4-(1,methyl-cyanomethyl)-aminobenzamide and4-Isothiocyanato-2-trifluoromethylbenzonitrile in DMF and heating toform a first mixture, and processing as above.

The invention also provides a method of synthesizing RD162″, comprisingmixing N-Methyl-2-fluoro-4-(1-cyanocyclopentyl)aminobenzamide,4-isothiocyanato-2-trifluoromethyl benzonitrile, and DMF and heatingunder reflux to form a first mixture, and processing as above.

The invention further provides a method of synthesizing RD169,comprising mixing N,N-Dimethyl4-[4-(1-cyanocyclobutylamino)phenyl]butanamide,4-isothiocyanato-2-trifluoromethyl benzonitrile, and DMF and heatingunder reflux to form a first mixture; and processing as above.

The invention provides a method of synthesizing RD170, comprising mixingDMSO, dichloromethane, and oxalyl chloride to form a first mixture,adding4-(4-(7-(4-Cyano-3-(trifluoromethyl)phenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)phenyl)butanamideto the first mixture to form a second mixture; adding triethylamine tothe second mixture to form a third mixture; warming the third mixtureand quenching with aqueous NH₄Cl to form a fourth mixture; extracting anorganic layer from the fourth mixture; and isolating the compound fromthe organic layer.

Further compounds according to the invention have the formula

wherein R5 is CN or NO2 or SO2R11, wherein R6 is CF3, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,halogenated alkyl, halogenated alkenyl, halogenated akynyl, halogen,wherein A is sulfur (S) or oxygen (O), wherein B is O or S or NR8,wherein R8 is selected from the group consisting of H, methyl, aryl,substituted aryl, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, arylalkyl, arylalkenyl,arylalkynyl, heterocyclic aromatic or non-aromatic, substitutedheterocyclic aromatic or non-aromatic, cycloalkyl, substitutedcycloalkyl, SO2R11, NR11R12, (CO)OR11, (CO)NR11R12, (CO)R11, (CS)R11,(CS)NR11R12, (CS)OR11,

wherein D is S or O and E is N or O and G is alkyl, aryl, substitutedalkyl or substituted aryl; or D is S or O and E-G together are C1-C4lower alkyl,

wherein R1 and R2 are independently alkyl, haloalkyl, hydrogen, aryl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, halogenated alkenyl, halogenated akynyl, arylalkyl,arylalkenyl, arylalkynyl, heterocylic aromatic or non-aromatic,substituted heterocyclic aromatic or non-aromatic, cycloalkyl,substituted cycloalkyl, or R1 and R2 are connected to form a cycle whichcan be heterocyclic, substituted heterocyclic, cycloalkyl, substitutedcycloalkyl,

wherein X is carbon or nitrogen and can be at any position in the ring,and

wherein R3, R4, and R7 are independently selected from the groupconsisting of hydrogen, halogen, methyl, methoxy, formyl, haloacetoxy,trifluoromethyl, cyano, nitro, hydroxyl, phenyl, amino, methylcarbamoyl,methylcarbamoyl-substituted alkyl, dimethylcarbamoyl-substituted alkyl,methoxycarbonyl, acetamido, methanesulfonamino, carbamoyl-substitutedalkyl, methanesulfonyl, 4-methanesulfonyl-1-piperazinyl, piperazinyl,hydroxyethylcarbamoyl-substituted alkyl, hydroxyl-substituted alkyl,hydroxyl-substituted alkenyl, carbamoyl-substituted alkenyl,methoxycarbonyl-substituted alkyl, cyano-substituted alkyl,

aryl, substituted aryl, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, halogenated alkenyl, halogenatedalkynyl, SO2R11, NR11R12, NR12(CO)OR11, NH(CO)NR11R12, NR12 (CO)R11,O(CO)R11, O(CO)OR11, O(CS)R11, NR12 (CS)R11, NH(CS)NR11R12, NR12(CS)OR11, arylalkyl, arylalkenyl, arylalkynyl, heterocyclic aromatic ornon-aromatic, substituted heterocyclic aromatic or non-aromatic,cycloalkyl, substituted cycloalkyl, haloalkyl,methylsulfonecarbamoyl-substituted alkyl, methylaminomethyl,dimethylaminomethyl, methylsulfonyloxymethyl, methoxycarbonyl,acetamido, methanesulfonamido, carbamoyl-substituted alkyl,carboxymethyl, methoxycarbonylmethyl, methanesulfonyl,4-cyano-3-trifluoromethylphenylcarbamoyl-substituted alkyl,carboxy-substituted alkyl, dimethyl ethoxy)carbonyl)-1-piperazinyl,hydroxyethylcarbamoyl-substituted alkyl,hydroxyethoxycarbonyl-substituted alkyl,3-cyano-4-trifluoromethylphenylcarbamoyl,

wherein R11 and R12 are independently hydrogen, aryl, aralkyl,substituted aralkyl, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, halogenated alkyl, halogenatedalkenyl, halogenated alkynyl, arylalkyl, arylalkenyl, arylalkynyl,heterocyclic aromatic or non-aromatic, substituted heterocyclic aromaticor non-aromatic, cycloalkyl, or substituted cycloalkyl, or R11 and R12can be connected to form a cycle which can be heterocyclic aromatic ornon-aromatic, substituted heterocyclic aromatic, cycloalkyl, orsubstituted cycloalkyl.

Such compounds have substantial androgen receptor antagonist activityand no substantial agonist activity on hormone refractory prostatecancer cells.

The invention encompasses a method comprising providing at least onesuch compound, measuring inhibition of androgen receptor activity forthe compound and determining if the inhibition is above a firstpredetermined level, measuring stimulation of androgen receptor activityin hormone refractory cancer cells for the compound and determining ifthe stimulation is below a second predetermined level, and selecting thecompound if the inhibition is above the first predetermined level andthe stimulation is below the second predetermined level. Thepredetermined levels may be those of bicalutamide. The step of measuringinhibition may comprise measuring inhibitory concentration (IC50) in anAR response reporter system or a prostate specific antigen secretingsystem. The step of measuring stimulation may comprise measuring foldinduction by increasing concentrations in an AR response reporter systemor a prostate specific antigen secreting system. The method of measuringinhibition and/or stimulation may comprise measuring an effect of thecompound on tumor growth in an animal.

BRIEF DESCRIPTION OF THE DRAWINGS

The following Figures present the results of pharmacological examinationof certain compounds.

FIG. 1 is a graph depicting that bicalutamide displays an agonisticeffect on LNCaP-AR. Agonistic activities of bicalutamide inAR-overexpressed hormone refractory prostate cancer. LNCaP cells withoverexpressed AR were treated with increasing concentrations of DMSO asvehicle or bicalutamide in the absence of R1881. Activities of ARresponse reporter were measured.

FIG. 2 is a graph depicting an antagonistic assay of bicalutamide onLNCaP-AR. Agonistic activities of bicalutamide in hormone sensitiveprostate cancer. LNCaP cells were treated with increasing concentrationsof DMSO as vehicle or bicalutamide in the absence of R1881. Activitiesof AR response reporter were measured.

FIG. 3 is a graph depicting the effect of compounds on LNCaP-AR.

FIG. 4 is a graph depicting the effect of compounds on LNCaP-AR.

FIG. 5 is a graph depicting the inhibition effect on LNCaP-AR.

In FIGS. 6-10, example 5-3b is RD7 and example 7-3b is RD37.

FIG. 6. Inhibition on growth of AR-overexpressed LNCaP cells. Androgenstarved LNCaP cells with overexpressed AR were treated with increasingconcentrations of DMSO as vehicle or test substances in the presence of100 pM of R1881. After 4 days of incubation, cell growth was measured byMTS assay.

FIG. 7. Inhibitory effect on growth of AR-overexpressed LNCaP xenograftmodel. Mice with established LN-AR xenograft tumors were randomized andtreated with indicated compounds orally once daily. Tumor size wasmeasured by caliber. (A), mice were treated with 1 mg per kg ofbicalutamide, example 7-3b, or vehicle for 44 days. (B), mice weretreated with vehicle, 0.1, 1, or 10 mg per kg of example 7-3b for 44days.

FIG. 8. Inhibitory effect on PSA expression of AR-overexpressed LNCaPxenograft model. Mice were treated with vehicle, 0.1, 1, or 10 mg per kgof example 7-3b for 44 days orally once daily. The tumors were taken outfrom the mice after 44 days of treatment, tumor lysate was extracted,and PSA level in tissue lysate was determined by ELISA.

FIG. 9. Inhibitory effect on growth and PSA of hormone refractory LAPC4xenograft model. Mice with established tumors were randomized andtreated with 1 mg per kg of bicalutamide, example 7-3b, or vehicle for17 days orally once daily. (A), tumor size was measured by caliber. (B),the tumors were taken out from the mice after 17 days of treatment,tumor lysate was extracted, and PSA level in tissue lysate wasdetermined by ELISA.

FIG. 10. Inhibitory effect on growth of hormone sensitive prostatecancer cells. Androgen starved LNCaP cells were treated with increasingconcentrations of DMSO as vehicle or test substances in the presence of1 μM of R1881. After 4 days of incubation, cell growth was measured byMTS assay.

FIG. 11 is a graph of tumor size. AR overexpressing LNCaP cells wereinjected in the flanks of castrated SOD mice, subcutaneously. Whentumors reached about 100 cubic mm, they were randomized into fivegroups. Each group had nine animals. After they reached this tumorvolume, they were given orally with either vehicle, bicalutamide orRD162 at 10 or 50 mg/kg everyday. The tumors were measuredthree-dimensionally, width, length and depth, using a caliper.

FIG. 12 depicts experimental results of tumor size. At day 18, theanimals were imaged via an optical CCD camera, 3 hours after last doseof treatment. A ROI was drawn over the tumor for luciferase activitymeasurement in photon/second. The right panels is a representation ofthe ROIs measurements.

FIG. 13 is a graph depicting the pharmacokinetic curves of RD162 fromintravenous (upper curve) and oral administration (lower curve).

FIG. 14 is a graph depicting PSA absorbance measured for LN-AR cellsafter treatment with various doses of several compounds.

FIG. 15 presents a table providing several characteristics of compounds.FIG. 15 also presents a graph providing the pharmacokineticcharacteristics of several compounds in terms of compound serumconcentration as a function of time.

FIG. 16 is a chart depicting prostate weight after treatment withvarious compounds. 10, 25, or 50 mg of compound per kilogram body weightwere administered per day, as indicated by the label of a bar. Thecompounds were administered to healthy FVB mice. After treatment withcompound for 14 days, the urogenital tract weight was determined byremoving and weighing the semi-vesicles, prostate, and bladder. Threemice were administered a given compound to obtain the data presented bya bar in the chart. A set of mice was not treated with a compound: dataare presented in the bar labeled “untreated”. Another set of mice wastreated only with vehicle solution: data are presented in the barlabeled “vehicle”.

FIG. 17 is a graph presenting a PSA assay performed along with theexperimental protocol presented in FIG. 6.

FIG. 18 is a graph presenting the effect of various dose regimens ofRD162 on tumor volume.

FIG. 19 is a graph presenting the rate of photon emission associatedwith luciferase activity at day 17 relative to the rate at day 0 aftertreatment with RD162 at doses of 0.1, 1, and 10 mg per kilogram bodyweight per day and without treatment with RD162.

FIG. 20 presents the results of an experiment in which SCID mice wereinjected with the LN-AR (HR) cell line to induce tumor growth. One setof mice were treated with the compound RD162 at a dose of 10 mg perkilogram body weight per day; the other set of mice were treated onlywith vehicle solution. (A) The relative tumor volume as a function oftime shown for each set of mice. (B) Images of each set of mice withphoton emission associated with luciferase activity at day 31 shown ascolor contours. (C) Rate of photon emission associated with luciferaseactivity shown at several times for each set of mice.

FIG. 21 is a graph presenting PSA absorbance associated with LN-AR cellstreated with various concentrations of RD162, RD162′, RD162″, and RD170and vehicle solution.

FIG. 22 is a graph presenting PSA absorbance associated with LN-CaPcells treated with various concentrations of RD37, RD131, RD162,bicalutamide, and DMSO.

FIG. 23 presents results of an experiment conducted with wild typenontransgenic mice (WT), castrated luciferase transgenic mice (Cast),and non-castrated luciferase transgenic mice (Intact). Data are shownfor castrated luciferase transgenic mice treated with an implantedtestosterone pellet yielding 12.5 mg per kilogram body weight with a 90day release period (T/Cast), and data are shown for non-castratedluciferase transgenic mice treated with an implanted testosterone pelletyielding 12.5 mg per kilogram body weight with a 90 day release period(Intact-FT). Data are shown for castrated luciferase transgenic micetreated with the implanted testosterone pellet and with bicalutamide(BIC+T/Cast) or with RD162 (RD162+T/Cast) at 10 mg per kilogram bodyweight per day. (A) Urogenital tract weight at 14 days. (B) Photonemission rate at 14 days. In all cases, a hormone refractory diseasestate was not induced.

FIG. 24 is a graph of luciferase activity of the L1AR cell line dosedwith various compounds administered at concentrations ranging from 125nmol to 1000 μmol.

FIG. 25 is a graph of luciferase activity for the LN/AR cell line forvarious compounds administered at concentrations ranging from 1.25 to 10μmol.

FIG. 26 is a graph of luciferase activity for the 4AR cell line forvarious compounds administered at concentrations ranging from 1.25 to 10μmol.

FIG. 27 is a graph of PSA levels for the 1AR cell line for variouscompounds administered at concentrations ranging from 1.25 to 10 μmol.

FIG. 28 is a graph of PSA levels for the LN/AR cell line for variouscompounds administered at concentrations ranging from 125 nmol to 1000μmol.

FIG. 29 is a graph of luciferase activity for various compoundsadministered at concentrations ranging from 125 nmol to 1000 μmol.

DETAILED DESCRIPTION

Embodiments of the invention are discussed in detail below. Indescribing embodiments, specific terminology is employed for the sake ofclarity. However, the invention is not intended to be limited to thespecific terminology so selected. A person skilled in the relevant artwill recognize that other equivalent parts can be employed and othermethods developed without parting from the spirit and scope of theinvention. All references cited herein are incorporated by reference asif each had been individually incorporated.

Synthesis of Diarylhydantoin Compounds

The invention provides for synthesis of diarylthiohydantoin compoundhaving the formula

with R71 including an alkyl chain of from 1 to 4 carbon atoms. Forexample, R72 can be carbamoyl, e.g., —(CO)NH₂, or methylcarbamoyl, e.g.,—(CO)NHCH₃. An amide group bonded at the carbon atom of the carbonyl toanother structure is termed a carbamoyl substituent. For example, R73can be a fluorine or a hydrogen atom. That is, a fluorine atom can beattached to any one of the carbons of the right-hand aryl ring which arenot bonded to the R72 substituent or the nitrogen atom. Alternatively,no fluorine atom can be attached to the carbons of the right-hand arylring which are not bonded to the R72 substituent or the nitrogen atom.For example, a hydrogen atom can be attached to each of the carbons ofthe right-hand aryl ring which are not bonded to the R72 substituent orthe nitrogen atom.

For example, as further presented below (see, for example, FIGS. 3, 5,11-13), the compound having the formula

exhibited surprisingly potent antagonistic activities with minimalagonistic activities for overexpressed AR in hormone refractory prostatecancer.

A list of several compounds according to this invention is presented inTables 5-11. The compounds are grouped into tiers, with Tier 1 to Tier 3compounds being expected to be superior to bicalutamide for thetreatment of prostate cancer, Tier 4 compounds being comparable tobicalutamide in effectiveness, and Tier 5 and Tier 6 compounds beingworse than bicalutamide for the treatment of prostate cancer. A moredetailed description of the protocol used to rank the compounds intotiers is presented below.

DEFINITIONS

As used herein, the term “alkyl” denotes branched or unbranchedhydrocarbon chains, preferably having about 1 to about 8 carbons, suchas, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl,tert-butyl, 2-methylpentyl pentyl, hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl and the like. “Substituted alkyl”includes an alkyl group optionally substituted with one or morefunctional groups which may be attached to such chains, such as,hydroxyl, bromo, fluoro, chloro, iodo, mercapto or thio, cyano,alkylthio, heterocyclyl, aryl, heteroaryl, carboxyl, carbalkoyl, alkyl,alkenyl, nitro, amino, alkoxyl, amido, and the like to form alkyl groupssuch as trifluoro methyl, 3-hydroxyhexyl, 2-carboxypropyl,2-fluoroethyl, carboxymethyl, cyanobutyl and the like.

Unless otherwise indicated, the term “cycloalkyl” as employed hereinalone or as part of another group includes saturated or partiallyunsaturated (containing 1 or more double bonds) cyclic hydrocarbongroups containing 1 to 3 rings, including monocyclicalkyl, bicyclicalkyland tricyclicalkyl, containing a total of 3 to 20 carbons forming therings, preferably 3 to 10 carbons, forming the ring and which may befused to 1 or 2 aromatic rings as described for aryl, which includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl, cyclodecyl and cyclododecyl, cyclohexenyl. “Substitutedcycloalkyl” includes a cycloalkyl group optionally substituted with 1 ormore substituents such as halogen, alkyl, alkoxy, hydroxy, aryl,aryloxy, arylalkyl, cycloalkyl, alkylamido, alkanoylamino, oxo, acyl,arylcarbonylamino, amino, nitro, cyano, thiol and/or alkylthio and/orany of the substituents included in the definition of “substitutedalkyl.” For example,

and the like.

Unless otherwise indicated, the term “alkenyl” as used herein by itselfor as part of another group refers to straight or branched chainradicals of 2 to 20 carbons, preferably 2 to 12 carbons, and morepreferably 2 to 8 carbons in the normal chain, which include one or moredouble bonds in the normal chain, such as vinyl, 2-propenyl, 3-butenyl,2-butenyl, 4-pentenyl, 3-pentenyl, 2-hexenyl, 3-hexenyl, 2-heptenyl,3-heptenyl, 4-heptenyl, 3-octenyl, 3-nonenyl, 4-decenyl, 3-undecenyl,4-dodecenyl, 4,8,12-tetradecatrienyl, and the like. “Substitutedalkenyl” includes an alkenyl group optionally substituted with one ormore substituents, such as the substituents included above in thedefinition of “substituted alkyl” and “substituted cycloalkyl.”

Unless otherwise indicated, the term “alkynyl” as used herein by itselfor as part of another group refers to straight or branched chainradicals of 2 to 20 carbons, preferably 2 to 12 carbons and morepreferably 2 to 8 carbons in the normal chain, which include one or moretriple bonds in the normal chain, such as 2-propynyl, 3-butynyl,2-butynyl, 4-pentynyl, 3-pentynyl, 2-hexynyl, 3-hexynyl, 2-heptynyl,3-heptynyl, 4-heptynyl, 3-octynyl, 3-nonynyl, 4-decynyl, 3-undecynyl,4-dodecynyl and the like. “Substituted alkynyl” includes an alkynylgroup optionally substituted with one or more substituents, such as thesubstituents included above in the definition of “substituted alkyl” and“substituted cycloalkyl.”

The terms “arylalkyl”, “arylalkenyl” and “arylalkynyl” as used alone oras part of another group refer to alkyl, alkenyl and alkynyl groups asdescribed above having an aryl substituent. Representative examples ofarylalkyl include, but are not limited to, benzyl, 2-phenylethyl,3-phenylpropyl, phenethyl, benzhydryl and naphthylmethyl and the like.“Substituted arylalkyl” includes arylalkyl groups wherein the arylportion is optionally substituted with one or more substituents, such asthe substituents included above in the definition of “substituted alkyl”and “substituted cycloalkyl.”

The terms “arylalkyl”, “arylalkenyl” and “arylalkynyl” as used alone oras part of another group refer to alkyl, alkenyl and alkynyl groups asdescribed above having an aryl substituent. Representative examples ofarylalkyl include, but are not limited to, benzyl, 2-phenylethyl,3-phenylpropyl, phenethyl, benzhydryl and naphthylmethyl and the like.“Substituted arylalkyl” includes arylalkyl groups wherein the arylportion is optionally substituted with one or more substituents, such asthe substituents included above in the definition of “substituted alkyl”and “substituted cycloalkyl.”

The term “halogen” or “halo” as used herein alone or as part of anothergroup refers to chlorine, bromine, fluorine, and iodine.

The terms “halogenated alkyl”, “halogenated alkenyl” and “alkynyl” asused herein alone or as part of another group refers to “alkyl”,“alkenyl” and “alkynyl” which are substituted by one or more atomsselected from fluorine, chlorine, bromine, fluorine, and iodine.

Unless otherwise indicated, the term “aryl” or “Ar” as employed hereinalone or as part of another group refers to monocyclic and polycyclicaromatic groups containing 6 to 10 carbons in the ring portion (such asphenyl or naphthyl including 1-naphthyl and 2-naphthyl) and mayoptionally include one to three additional rings fused to a carbocyclicring or a heterocyclic ring (such as aryl, cycloalkyl, heteroaryl orcycloheteroalkyl rings).

“Substituted aryl” includes an aryl group optionally substituted withone or more functional groups, such as halo, haloalkyl, alkyl,haloalkyl, alkoxy, haloalkoxy, alkenyl, trifluoromethyl,trifluoromethoxy, alkynyl, cycloalkyl-alkyl, cycloheteroalkyl,cycloheteroalkylalkyl, aryl, heteroaryl, arylalkyl, aryloxy,aryloxyalkyl, arylalkoxy, alkoxycarbonyl, arylcarbonyl, arylalkenyl,aminocarbonylaryl, arylthio, arylsulfinyl, arylazo, heteroarylalkyl,heteroarylalkenyl, heteroarylheteroaryl, heteroaryloxy, hydroxy, nitro,cyano, amino, substituted amino wherein the amino includes 1 or 2substituents (which are alkyl, aryl or any of the other aryl compoundsmentioned in the definitions), thiol, alkylthio, arylthio,heteroarylthio, arylthioalkyl, alkoxyarylthio, alkylcarbonyl,arylcarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl,aminocarbonyl, alkylcarbonyloxy, arylcarbonyloxy, alkylcarbonylamino,arylcarbonylamino, arylsulfinyl, arylsulfinylalkyl, arylsulfonylamino orarylsulforiaminocarbonyl and/or any of the alkyl substituents set outherein.

Unless otherwise indicated, the term “heterocyclic” or “heterocycle”, asused herein, represents an unsubstituted or substituted stable 5- to10-membered monocyclic ring system which may be saturated orunsaturated, and which consists of carbon atoms and from one to fourheteroatoms selected from N, O or S, and wherein the nitrogen and sulfurheteroatoms may optionally be oxidized, and the nitrogen heteroatom mayoptionally be quaternized. The heterocyclic ring may be attached at anyheteroatom or carbon atom which results in the creation of a stablestructure. Examples of such heterocyclic groups include, but is notlimited to, piperidinyl, piperazinyl, oxopiperazinyl, oxopiperidinyl,oxopyrrolidinyl, oxoazepinyl, azepinyl, pyrrolyl, pyrrolidinyl, furanyl,thienyl, pyrazolyl, pyrazolidinyl, imidazolyl, imidazolinyl,imidazolidinyl, pyridyl, pyrazinyl, pyridazinyl, oxazolyl, oxazolidinyl,isooxazolyl, isoxazolidinyl, morpholinyl, thiazolyl, thiazolidinyl,isothiazolyl, thiadiazolyl, tetrahydropyranyl, thiamorpholinyl,thiamorpholinyl sulfoxide, thiamorpholinyl sulfone, and oxadiazolyl. Theterm “heterocyclic aromatic” as used here in alone or as part of anothergroup refers to a 5- or 7-membered aromatic ring which includes 1, 2, 3or 4 hetero atoms such as nitrogen, oxygen or sulfur and such ringsfused to an aryl, cycloalkyl, heteroaryl or heterocycloalkyl ring (e.g.benzothiophenyl, indolyl), and includes possible N-oxides. “Substitutedheteroaryl” includes a heteroaryl group optionally substituted with 1 to4 substituents. such as the substituents included above in thedefinition of “substituted alkyl” and “substituted cycloalkyl.” Examplesof heteroaryl groups include the following:

and the like.

Example 1 4-isothiocyanato-2-trifluoromethylbenzonitrile, (1a)

4-Amino-2-trifluoromethylbenzonitrile, (2.23 g, 12 mmol) was addedportionwise over 15 minutes into the well-stirred heterogeneous mixtureof thiophosgene (1 ml, 13 mmol) in water (22 ml) at room temperature.Stirring was continued for an additional 1 h. The reaction medium wasextracted with chloroform (3×15 ml). The combined organic phase wasdried over MgSO₄ and evaporated to dryness under reduced pressure toyield desired product, 4-isothiocyanato-2-trifluoromethylbenzonitrile,(1a), as brownish solid and was used as such for the next step (232 g,11.9 mmol, 99%).

Example 2 2-1). (4-aminophenyl)carbamic acid tert-butyl ester, (2a)

An aqueous solution of potassium carbonate (1.52 g, 11 mmol in 5 ml ofwater) was added to a solution of 1,4-diaminobenzene (3.24 g, 30 mmol)in THF (30 ml) and DMF (10 ml). To this mixture was added di-tert-butylpyrocarbonate, Boc₂O (2.18 g, 10 mmol), dropwise over 0.5 h. Thereaction mixture was stirred for an additional 4 h at room temperature.The mixture was then poured into cold water (40 ml) and extracted withchloroform (3×50 ml). The combined organic phase was dried over MgSO₄and concentrated to yield a brown residue which was subjected to flashchromatography (dichloromethane/acetone, 4:1) to afford(4-aminophenyl)carbamic acid tert-butyl ester, (2a) as a yellow solid(1.98 g, 9.5 mmol, 95%) (yield based on Boc₂O).

2-2). {4-[(1-cyano-1-methylethyl)amino]phenyl}carbamic acid ten-butylester, 2b

The mixture of 2a (0.83 g, 4 mmol), acetone cyanohydrin (4 ml) and MgSO₄(2 g) was heated to 80° C. and stirred over 2.5 h. After cooling down toroom temperature, compound 2b was crystallized into water (30 ml). Thesolid was filtered and dried to yield{4-[(1-cyano-1-methylethyl)amino]phenyl}carbamic acid tert-butyl ester,2b (1.08 g, 3.9 mmol, 98%).

2-3).{4-[3-(4-cyano-3-trifluoromethylphenyl)-4-imino-5,5-dimethyl-2-thioxo-imidazolidin-1-yl]phenyl}carbamicacid tert-butyl ester, (2c)

Triethylamine (0.202 g, 2 mmol) was added to a solution of 1a (0.456 g,2 mmol) and 2b (0.57 g, 2 mmol) in dry THF (5 ml). The reaction mixturewas stirred at room temperature for 15 h and then concentrated to yielda dark residue which was subjected to flash chromatography (ethylether/acetone, 97:3) to afford(4-[3-(4-cyano-3-trifluoromethylphenyl)-4-imino-5,5-dimethyl-2-thioxo-imidazolidin-1-yl]phenyl)carbamicacid tert-butyl ester, (2c) (0.15 g, 0.3 mmol, 15%).

2-4).4-[3-(4-aminophenyl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile,2d, [14D9]

The mixture of 2c (0.15 g, 0.3 mmol) in HCl aq, 3N. (1 ml) and methanol(4 ml) was heated to reflux for 2 h. After being cooled to roomtemperature, the reaction mixture was poured into cold water (5 ml) andextracted with dichloromethane (8 ml). The organic layer was dried overMgSO₄, concentrated and chromatographed (dichloromethane/acetone, 9:1)to yield4-[3-(4-aminophenyl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile,2d, [RD9] (0.118 g, 0.29 mmol, 97%) as a yellow solid.

¹H NMR (400 MHz, CDCl₃) δ 1.54 (s, 6H), 6.73-6.75 (m, 2H), 7.00-7.03 (m,2H), 8.02 (dd, J=8.2 Hz, J₂=1.8 Hz, 1H), 8.16 (d, J=1.8 Hz, 1H), 8.20(d, J=8.2 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 22.7, 66.2, 109.1, 114.3,114.9, 120.4, 122.0 (q, J=272.5 Hz), 127.0 (q, J=4.9 Hz), 130.4, 132.5(q, J=33.0 Hz), 133.4, 135.6, 138.5, 149.2, 175.3, 180.4.

2-5).4-[3-(4-azidophenyl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl]-2-tritluoromethylbenzonitrile,2e, [RD10]

An aqueous solution of sulfuric acid (25% wt, 1 ml) was added to asolution of 2d (0.10 g, 0.25 mmol) in acetone (1 ml) at −5° C. Anaqueous solution of NaNO₂ (0.024 g, 0.35 mmol, in 0.5 ml of water) wasadded slowly the above mixture over 0.1 h. The reaction mixture wasallowed to stir at −5° C. for an additional 1 h and then an aqueoussolution of NaN₃ (0.02 g, 0.3 mmol in 0.3 ml of water) was addeddropwise. Upon completion of the addition, the reaction medium waswarmed to room temperature and stirred for an additional 3 h. Theproduct was extracted with dichloromethane (3×5 ml). The combinedorganic layer was dried over MgSO₄, concentrated and chromatographed(dichloromethane) to yield4-[3-(4-azidophenyl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile,2e, [RD10] (0.08 g, 0.18 mmol, 72%) as a yellowish solid.

¹H NMR (400 MHz, CDCl₃) δ 1.54 (s, 6H), 7.17-7.20 (m, 2H), 7.27-7.30 (m,2H), 7.84 (dd, J=8.3 Hz, =1.8 Hz, 1H), 7.96 (d, J=1.8 Hz, 1H), 7.97 (d,J=8.3 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 23.7, 66.4, 110.1, 114.8,120.4, 122.1 (q, J=272.5 Hz), 127.0 (q, J=4.7 Hz), 131.1, 131.5, 132.3,133.3 (q, J=33.0 Hz), 135.3, 137.1, 141.7, 174.8, 180.1. MS forC₁₉H₁₃F₃N₆OS, calculated 430.4. found 430.1.

Example 3 3-1). 2-(4-hydroxyphenylamino)-2-methylpropanenitrile, 3a

A mixture of 4-aminophenol (1.09 g, 10 mmol), acetone cyanohydrin (10ml) and MgSO4 (2 g) was heated to 80° C. and stirred for 4 h. Afterconcentration of the medium under vacuum, compound 3a was crystallizedfrom water (20 ml). The solid was filtered and dried to yield2-(4-hydroxyphenylamino)-2-methylpropanenitrile, 3a (1.69 g, 9.6 mmol,96%).

3-2).4-[3-(4-hydroxyphenyl)-5-imino-4,4-dimethyl-2-thioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile,3b

Triethylamine (0.101 g, 1 mmol) was added to a solution of 1a (0.456 g,2 mmol) and 3a (0.352 g, 2 mmol) in dry THF (5 ml). The reaction mixturewas stirred at 0° C. for 48 h and then concentrated to yield a darkresidue which was subjected to flash chromatography(dichloromethane/acetone, 85:15) to afford4-[3-(4-hydroxyphenyl)-5-imino-4,4-dimethyl-2-thioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile,3b (0.274 g, 0.68 mmol, 34%).

3-3).4-[3-(4-hydroxyphenyl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile,3c, [RD8]

A mixture of 3b (0.202 g, 0.5 mmol) in HCl aq., 2N (2 ml) and methanol(5 ml) was heated to reflux for 2 h. After being cooled to roomtemperature, the reaction mixture was poured into cold water (10 ml) andextracted with ethyl acetate (10 ml). The organic layer was dried overMgSO₄, concentrated and chromatographed (dichloromethane/acetone, 9:1)to yield4-[3-(4-hydroxyphenyl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile,3c, [RD8] (0.198 g, 0.49 mmol, 98%) as a white powder.

¹H NMR (CDCl₃, 400 MHz) δ1.57 (s, 6H), 6.26 (s, OH), 6.90-6.93 (m, 2H),7.11-7.14 (m, 2H), 7.84 (dd, J₁=8.3 Hz, J₂=1.8 Hz, 1H), 7.95-7.98 (m,2H); ¹³C NMR (CDCl₃,100 MHz) δ 23.6, 66.5, 109.9, 114.9, 115.7, 116.8,121.9 (q, J=272.7 Hz), 127.2 (q, J=4.7 Hz), 130.6, 132.3, 133.5 (q,J=33.2 Hz), 135.3, 137.2, 157.0, 175.3, 180.2.

Example 4 Chloroacetic acid4-[3-(4-cyano-3-trifluoromethylphenyl)-5,5-dimethyl-4-oxo-2-thioxoimidazolidin-1-yl]phenylester, 4a, [RD13]

Chloroacetyl chloride (0.045 g, 0.4 mmol) was added to a mixture of 3c(0.101 g, 0.25 mmol) and triethylamine (0.041 g, 0.41 mmol) in dry THF(1.5 ml). The mixture was stirred at room temperature for 4 h.Triethylamine hydrochloride was filtered off. The filtrate wasconcentrated and chromatographed (dichloromethane/acetone, 95:5) toyield 84% of Chloroacetic acid4-[3-(4-cyano-3-trifluoromethylphenyl)-5,5-dimethyl-4-oxo-2-thioxoimidazolidin-1-yl]phenylester, 4a, [RD13] (0.101 g, 0.21 mmol) as white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.58 (s, 6H), 4.32 (s, 2H), 7.33 (s, 4H), 7.83(dd, J=8.3 Hz, J₂=1.9 Hz, 1H), 7.95-7.97 (m, 2H); ¹³C NMR (CDCl₃,100MHz) δ 23.7, 40.8, 66.5, 110.1, 114.8, 121.9 (q, J=272.5 Hz), 122.7,127.1 (q, J=4.7 Hz), 130.9, 132.3, 132.9, 133.5 (q, J=33.2 Hz), 135.3,137.1, 150.9, 165.5, 174.8, 180.0.

Example 5 5-1a). 2-methyl-2-(4-methylphenyl)aminopropanenitrile, 5a

A mixture of p-toluidine (1.07 g, 10 mmol) and acetone cyanohydrin (10ml) was heated to 80° C. and stirred for 4 h. The medium wasconcentrated and dried under vacuum to yield2-methyl-2-(4-methylphenyl)aminopropanenitrile, 5a (1.72 g, 9.9 mmol,99%) as brown solid.

5-1b). 2-methyl-2-(4-methylphenyl)aminopropanenitrile, 5a

Sodium cyanide (0.735 g, 15 mmol) was added to a mixture of p-toluidine(1.07 g, 10 mmol) and acetone (1.16 g, 20 mmol) in 90% acetic acid (10ml). The reaction mixture was stirred at room temperature for 12 h andthen ethyl acetate (50 ml) was added. The organic layer was washed withwater (4×30 ml), dried over magnesium sulfate and concentrated undervacuum to dryness to yield2-methyl-2-(4-methylphenyl)aminopropanenitrile, 5a (1.65 g, 9.5 mmol,95%) as a brown solid.

5-2).4-[3-(4-methylphenyl)-5-imino-4,4-dimethyl-2-thioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile,5b

Triethylamine (0.101 g, 1 mmol) was added to a solution of 1a (0.456 g,2 mmol) and 5a (0.348 g, 2 mmol) in dry THF (3 ml). The reaction mixturewas stirred at 0° C. for 2 days and then concentrated to yield a darkresidue which was subjected to flash chromatography(dichloromethane/acetone, 95:5) to afford4-[3-(4-methylphenyl)-5-imino-4,4-dimethyl-2-thioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile,5b (0.136 g, 0.34 mmol, 17%).

5-3a).4-[3-(4-methylphenyl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile,5c

A mixture of 5b (0.121 g, 0.3 mmol) in HCl aq., 2N (2 ml) and methanol(5 ml) was heated to reflux for 2 h. After being cooled to roomtemperature, the reaction mixture was poured into cold water (10 ml) andextracted with ethyl acetate (10 ml). The organic layer was dried overMgSO₄, concentrated and chromatographed (dichloromethane) to yield4-[3-(4-methylphenyl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile,5c (0.118 g, 0.294 mmol, 98%) as a white powder.

5-3b).4-[3-(4-methylphenyl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl]-2-trifluoromethyl-benzonitrile,5c, [RD7]

A mixture of 1a (0.547 g, 2.4 mmol) and 5a (0.348 g, 2 mmol) in dry DMF(0.6 ml) was stirred for 36 h. To this mixture were added methanol (20ml) and 2N HCl (5 ml). The second mixture was refluxed for 6 h. Afterbeing cooled to room temperature, the reaction mixture was poured intocold water (30 ml) and extracted with ethyl acetate (40 ml). The organiclayer was dried over MgSO₄, concentrated and chromatographed(dichloromethane) to yield4-[3-(4-methylphenyl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl]-2-trifluoromethyl-benzonitrile,5c, [RD7] (0.596 g, 1.48 mmol, 74%) as a white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.61 (s, 6H), 2.44 (s, 3H), 7.17-7.20 (m, 2H),7.33-7.36 (m, 2H), 7.86 (dd, J_(J)=8.3 Hz, J₂=1.8 Hz, 1H), 7.96-7.98 (m,2H); ¹³C NMR (CDCl₃, 100 MHz) δ 21.3, 23.6, 66.4, 110.0, 114.9, 121.9(q, J=272.6 Hz), 127.1 (q, J=4.7 Hz), 129.2, 130.6, 132.2, 132.3, 133.4(q, J=33.2 Hz), 135.2, 137.2, 140.1, 175.1, 179.9.

Example 6 6-1). 2-methyl-2-phenylaminopropanenitrile, 6a

A mixture of aminobenzene (0.931 g, 10 mmol) and acetone cyanohydrin (2ml) was heated to reflux and stirred for 20 h. After being cold to roomtemperature, the reaction mixture was poured into ethyl acetate (40 ml)and washed with cold water (2×30 ml). The organic layer was dried overMgSO₄, concentrated under vacuum to dryness to yield2-methyl-2-phenylaminopropanenitrile, 6a (1.51 g, 9.4 mmol, 94%) asslurry brown liquid.

6-2).4-[3-phenyl-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile,6b, [RD10]

A mixture of 1a (0.274 g, 1.2 mmol) and 6a (0.160 g, 1 mmol) in dry DMF(0.2 ml) was stirred for 48 h. To this mixture were added methanol (10ml) and 2N HCl (3 ml). The second mixture was refluxed for 6 h. Afterbeing cooled to room temperature, the reaction mixture was poured intocold water (20 ml) and extracted with ethyl acetate (20 ml). The organiclayer was dried over MgSO₄, concentrated and chromatographed(dichloromethane) to yield4-[3-phenyl-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile,6b, [RD10] (0.276 g, 0.71 mmol, 71%) as a white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.60 (s, 6H), 7.28-7.31 (m, 2H), 7.50-7.58 (m,3H), 7.85 (dd, =8.3 Hz, J2=1.8 Hz, 1H), 7.96-7.99 (m, 2H); ¹³C NMR(CDCl₃, 100 MHz) δ 23.7, 66.4, 110.2, 114.8, 121.9 (q, J=272.6 Hz),127.1 (q, J=4.7 Hz), 129.5, 129.8, 129.9, 132.2, 133.4 (q, J=33.2 Hz),135.1, 135.2, 137.2, 175.0, 179.9.

Example 7 7-1a). 1-(4-methylphenyl)aminocyclobutanenitrile, 7a

Sodium cyanide (0.147 g, 3 mmol) was added to a mixture of p-toluidine(0.214 g, 2 mmol) and cyclobutanone (0.21 g, 3 mmol) in 90% acetic acid(3 ml). The reaction mixture was stirred at room temperature for 12 hand then 20 ml of ethyl acetate was added. The organic layer was washedwith water (3×10 ml), dried over magnesium sulfate and concentratedunder vacuum to dryness to yield1-(4-methylphenypaminocyclobutanenitrile, 7a (0.343 g, 1.84 mmol, 92%)as a brown solid.

7-1b). 1-(4-methylphenyl)aminocyclobutanenitrile, 7a

Trimethylsilyl cyanide (0.93 ml, 7 mmol) was added dropwise to a mixtureof p-toluidine (0.535 g, 5 mmol) and cyclobutanone (0.42 g, 6 mmol). Thereaction mixture was stirred at room temperature for 6 h and thenconcentrated under vacuum to obtain a brown liquid which was subjectedto chromatography (dichloromethane) to yield1-(4-methylphenyl)aminocyclobutanenitrile, 7a (0.912 g, 4.9 mmol, 98%)as a yellowish solid.

7-2).4-(8-imino-6-thioxo-5-(4-methylphenyl)-5,7-diazaspiro[3.4]oct-7-yl)-2-trifluoromethylbenzonitrile,7b

To a solution of 1a (2.28 g, 10 mmol) in dry DMF (3 ml) was addedprogressively, over 20 hours, a solution of 7a (1.764 g, 9 mmol) in dryDMF (3 ml) at room temperature. The medium was stirred for an additional4 h. After DMF being evaporated, the residue was chromatographed(dichloromethane/acetone, 95:5) to afford4-(8-imino-6-thioxo-5-(4-methylphenyl)-5,7-diazaspiro[3.4]oct-7-yl)-2-trifluoromethylbenzonitrile,7b (1.937 g, 4.68 mmol, 52%).

7-3a).4-(8-oxo-6-thioxo-5-(4-methylphenyl)-5,7-diazaspiro[3.4]oct-7-yl)-2-trifluoromethylbenzonitrile,7c [RD37]

A mixture of 7b (0.041 g, 0.1 mmol) in HCl aq., 2N (3 ml) and methanol(1 ml) was heated to reflux for 2 h. After being cooled to roomtemperature, the reaction mixture was poured into cold water (5 ml) andextracted with ethyl acetate (6 ml). The organic layer was dried overMgSO₄, concentrated and chromatographed (dichloromethane) to yield4-(8-oxo-6-thioxo-5-(4-methylphenyl)-5,7-diaztspiro[3.4]oct-7-yl)-2-trifluoromethylbenzonitrite,4-(8-oxo-6-thioxo-5-(4-methylphenyl)-5,7-diazaspiro[3.4]oct-7-yl)-2-trifluoromethylbenzonilrile,7c (0.04 g, 0.096 mmol, 96%) as a white powder.

7-3b).4-(8-oxo-6-thioxo-5-(4-methylphenyl)-5,7-diazaspiro[3.4]oct-7-yl)-2-trifluoromethylbenzonitrile,7c, [RD37]

A mixture of 1a (0.912 g, 4 mmol) and 7a (0.558 g, 3 mmol) in dry DMF(0.5 ml) was stirred at room temperature for 24 h. To this mixture wereadded methanol (30 ml) and HCl aq. 2N (6 ml). The second mixture wasrefluxed for 6 h. After being cooled to room temperature, the reactionmixture was poured into cold water (50 ml) and extracted with ethylacetate (60 ml). The organic layer was dried over MgSO₄, concentratedand chromatographed (dichloromethane) to yield4-(8-oxo-6-thioxo-5-(4-methylphenyl)-5,7-diazaspiro[3.4]oct-7-yl)-2-trifluoromethylbenzonitrile,7c (0.959 g, 2.31 mmol, 77%) as a white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.62-1.69 (m, 1H), 2.16-2.22 (m, 1H), 2.46 (s,3H), 2.55-2.66 (m, 4H), 7.19-7.26 (m, 2H), 7.36-7.42 (m, 2H), 7.86 (dd,J₁=8.3 Hz, J₂=1.8 Hz, 1H), 7.96 (d, J=8.3 Hz, 1H), 7.99 (d, J=1.8 Hz,1H); ¹³C NMR (CDCl₃, 100 MHz) δ 13.7, 21.3, 31.4, 67.4, 109.9, 114.9,121.9 (q, J=272.6 Hz), 127.1 (q, J=4.7 Hz), 129.5, 130.8, 132.2, 132.4,133.3 (q, J=33.2 Hz), 135.2, 137.3, 140.1, 175.0, 180.0.

Example 8 8-1). 1-(4-methylphenyl)aminocyclopentanenitrile, 8a

Trimethylsilyl cyanide (0.865 ml, 7 mmol) was added dropwise to amixture of p-toluidine (0.535 g, 5 mmol) and cyclopentanone (0.589 g, 7mmol). The reaction mixture was stirred at room temperature for 6 h andthen concentrated under vacuum to obtain a brown liquid which wassubjected to chromatography (dichloromethane) to yield1-(4-methylphenyl)aminocyclopentanenitrile, 8a (0.981 g, 4.9 mmol, 98%)as a yellowish solid.

8-2).4-(4-oxo-2-thioxo-1-(4-methylphenyl)-1,3-diazaspiro[4.4]non-3-yl)-2-trifluoromethylbenzonitrile,8b, [RD35]

A mixture of 1a (0.296 g, 1.3 mmol) and 8a (0.2 g, 1 mmol) in dry DMF(0.2 ml) was stirred for 48 h. To this mixture were added methanol (10ml) and HCl aq. 2N (3 ml). The second mixture was refluxed for 6 h.After being cooled to room temperature, the reaction mixture was pouredinto cold water (20 ml) and extracted with ethyl acetate (30 ml). Theorganic layer was dried over MgSO₄, concentrated and chromatographed(dichloromethane) to yield4-(4-Oxo-2-thioxo-1-(4-methylphenyl)-1,3-diazaspiro[4.4]non-3-yl)-2-trifluoromethylbenzonitrile,8b, [RD35] (0.3 g, 0.7 mmol, 70%) as a white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.47-1.57 (m, 2H), 1.81-1.92 (m, 2H),2.20-2.24 (m, 2H), 2.27-2.34 (m, 2H), 2.43 (s, 3H), 7.18-7.22 (m, 2H),7.33-7.36 (m, 2H), 7.86 (dd, J=8.2 Hz, J₂=1.8 Hz, 1H), 7.96 (d, J=8.2Hz, 1H), 7.98 (d, J=1.8 Hz, 1H); ¹³C NMR (CDCl₃, 100 MHz) δ 21.3, 25.2,36.3, 75.1, 110.0, 114.9, 121.9 (q, J=272.5 Hz), 127.1 (q, J=4.7 Hz),129.5, 130.7, 123.2, 133.0, 133.4 (q, J=33.2 Hz), 135.1, 137.4, 140.0,176.3, 180.2.

Example 9 9-1). 1-(4-methylphenyl)aminocyclohexanenitrile, 9a

Sodium cyanide (0.147 g, 3 mmol) was added to a mixture of p-toluidine(0.214 g, 2 mmol) and cyclohexanone (0.294 g, 3 mmol) in acetic acid 90%(3 ml). The reaction mixture was stirred at room temperature for 12 hand then 20 ml of ethyl acetate was added. The organic layer was washedwith water (3×10 ml), dried over magnesium sulfate and concentratedunder vacuum to dryness to yield1-(4-methylphenyl)aminocyclohexanenitrile, 9a (0.398 g, 1.86 mmol, 93%)as a brown solid.

9-2).4-(4-imino-2-thioxo-1-(4-methylphenyl)-1,3-diazaspiro[4.5]dec-3-yl)-2-trifluoromethylbenzonitrile,9b

Triethylamine (0.05 g, 0.5 mmol) was added to a solution of 1a (0.228 g,1 mmol) and 9a (0.214 g, 1 mmol) in dry THF (2 ml). The reaction mixturewas stirred at room temperature for 2 days and then concentrated toyield a dark residue which was subjected to flash chromatography(dichloromethane/acetone, 95:5) to afford4-(4-imino-2-thioxo-1-(4-methylphenyl)-1,3-diazaspiro[4.5]dec-3-yl)-2-trifluoromethylbenzonitrile,9b (0.035 g, 0.08 mmol, 8%).

9-3).4-(4-oxo-2-thioxo-1-(4-methylphenyl)-1,3-diazaspiro[4.5]dec-3-yl)-2-trifluoromethylbenzonitrile,9c, [RD48]

A mixture of 9b (0.035 g, 0.08 mmol) in HCl aq., 2N (1 ml) and methanol(3 ml) was heated to reflux for 2 h. After being cooled to roomtemperature, the reaction mixture was poured into cold water (5 ml) andextracted with ethyl acetate (6 ml). The organic layer was dried overMgSO₄, concentrated and chromatographed (dichloromethane) to yield4-(4-oxo-2-thioxo-1-(4-methylphenyl)-1,3-diazaspiro[4.5]dec-3-yl)-2-trifluoromethylbenzonitrile,9c, [RD48] (0.034 g, 0.076 mmol, 95%) as a white powder.

¹H NMR (CDCl₃, 400 MHz) (m, 141), 1.64-1.76 (m, 4H), 2.03-2.12 (m, 5H),2.44 (s, 3H), 7.12-7.15 (m, 2H), 7.33-7.36 (m, 2H), 7.85 (dd, J=8.2 Hz,J₂=1.8 Hz, 1H), 7.96 (d, J=8.3 Hz, 1H), 7.97 (d, J=1.8 Hz, 1H); ¹³C NMR(CDCl₃, 100 MHz) δ 20.7, 21.3, 24.0, 32.6, 67.4, 109.9, 114.9, 122.0 (q,J=272.5 Hz), 127.3 (q, J=4.6 Hz), 130.0, 130.5, 132.0, 132.5, 133.3 (q,J=33.2 Hz), 135.2, 137.3, 140.1, 174.1, 180.1.

Example 10 10-1). 1-(4-methylphenyl)aminocyclohexanenitrile, 10a

Sodium cyanide (0.147 g, 3 mmol) was added to a mixture of p-toluidine(0.214 g, 2 mmol) and cycloheptanone (0.337 g, 3 mmol) in acetic acid90% (3 ml). The reaction mixture was stirred at room temperature for 12h and then 20 ml of ethyl acetate was added. The organic layer waswashed with water (3×10 ml), dried over magnesium sulfate andconcentrated under vacuum to dryness to yield1-(4-methylphenyl)aminocyclohexanenitrile, 10a (0.438 g, 1.92 mmol, 96%)as a brown solid.

10-2).4-(4-imino-2-thioxo-1-(4-methylphenyl)-1,3-diazaspiro[4.5]undec-3-yl)-2-trifluoromethylbenzonitrile,10b

Triethylamine (0.05 g, 0.5 mmol) was added to a solution of 1a (0.228 g,1 mmol) and 9a (0.228 g, 1 mmol) in dry THF (2 ml). The reaction mixturewas stirred at room temperature for 2 days and then concentrated toyield a dark residue which was subjected to flash chromatography(dichloromethane/acetone, 95:5) to afford4-(4-imino)-2-thioxo-1-(4-methylphenyl)-1,3-diazaspiro[4.5]undec-3-yl)-2-trifluoromethylbenzonitrile,10b (0.036 g, 0.08 mmol, 8%).

10-3).4-(4-oxo-2-thioxo-1-(4-methylphenyl)-1,3-diazaspiro[4.5]undec-3-yl)-2-trifluoromethylbenzonitrile,10c, [RD49]

A mixture of 9b (0.036 g, 0.08 mmol) in HCl aq., 2N (1 ml) and methanol(3 ml) was heated to reflux for 2 h. After being cooled to roomtemperature, the reaction mixture was poured into cold water (5 ml) andextracted with ethyl acetate (6 ml). The organic layer was dried overMgSO₄, concentrated and chromatographed (dichloromethane) to yield 10c(0.034 g, 0.075 mmol, 94%) as a white powder.

¹H NMR (CDCl₃, 400 MHz) δ1.24-134 (m, 2H), 1.37-1.43 (m, 2H), 133-1.60(m, 214), 1.74-1.82 (m, 2H), 2.19-2.25 (m, 4H), 2.44 (s, 3H), 7.16-7.19(m, 2H), 7.32-7.35 (m, 2H), 7.83 (dd, J=8.2 Hz, J_(Z)=1.8 Hz, 1H),7.95-7.97 (m, 2H); ¹³C NMR(CDCl₃, 100 MHz) δ 21.4, 22.2, 30.9, 36.3,71.1, 110.0, 114.9, 121.9 (q, J=272.5 Hz), 127.2 (q, J=4.6 Hz), 129.6,130.5, 132.3, 133.0, 133.2 (q, 0.7=33.2 Hz), 135.1, 137.4, 140.0, 175.9,179.7.

Example 11 11-1). 1-(4-hydroxyphenyl)aminocyclobutanenitrile, 11a

Trimethylsilyl cyanide (0.93 ml, 7 mmol) was added dropwise to a mixtureof 4-hydroxyaniline (0.545 g, 5 mmol) and cyclobutanone (0.42 g, 6mmol). The reaction mixture was stirred at room temperature for 6 h andthen concentrated under vacuum to obtain a brown liquid which wassubjected to chromatography (dichloromethane:acetone, 98:2) to yield 11a(0.903 g, 4.8 mmol, 96%) as a yellowish solid.

11-2).4-(8-oxo-6-thioxo-5-(4-hydroxyphenyl)-5,7-diazaspiro[3.4]oct-7-yl)-2-trifluoromethylbenzonitrile,11b, [RD58]

A mixture of 1a (0.57 g, 2.5 mmol) and 7a (0.376 g, 2 mmol) in dry DMF(0.5 ml) was stirred at room temperature for 40 h. To this mixture wereadded methanol (30 ml) and HCl aq. (5 ml). The second mixture wasrefluxed for 6 h. After being cooled to room temperature, the reactionmixture was poured into cold water (40 ml) and extracted with ethylacetate (50 ml). The organic layer was dried over MgSO₄, concentratedand chromatographed (dichloromethane:acetone, 98:2) to yield 11b (0.659g, 1.58 mmol, 79%) as a white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.55-1.63 (m, 1H), 2.01-2.09 (m, 1H),2.50-2.65 (m, 4H), 6.97-7.01 (m, 2H), 7.20-7.24 (m, 2H), 8.02 (dd,J₁=8.3 Hz, J₂=1.8 Hz, 1H), 8.14 (d, J=1.8 Hz, 1H), 8.21 (d, J=8.3 Hz,1H); ¹³C NMR (Acetone-d₆, 100 MHz) δ13.4, 31.3, 67.5, 108.9, 114.8,116.1, 123.5 (q, J=271.5 Hz), 127.4 (q, J=4.9 Hz), 131.3, 131.8 (q,J=32.7 Hz), 133.3, 135.5, 136.2, 138.5, 158.1, 175.1, 180.7.

Example 12 12-1). 1-(4-biphenylamino)cyclobutanecarbonitrile, 12a

Trimethylsilyl cyanide (0.2 ml, 1.5 mmol) was added dropwise to amixture of 4-biphenylamine (0.169 g, 1 mmol) and cyclobutanone (0.098 g,1.4 mmol). The reaction mixture was stirred at room temperature for 6 hand then concentrated under vacuum to obtain a brown liquid which wassubjected to chromatography (dichloromethane) to yield 12a (0.24 g, 0.97mmol, 97%) as a white solid.

12-2).4-(8-oxo-6-thioxo-5-(4-biphenyl)-5,7-diazaspiro[3.4]oct-7-yl)-2-trifluoromethylbenzonitrile,12b [RD57]

A mixture of 1a (0.137 g, 0.6 mmol) and 12a (0.124 g, 0.5 mmol) in dryDMF (0.2 ml) was stirred at room temperature for 3 days. To this mixturewere added methanol (5 ml) and HCl aq. 2N (1 ml). The second mixture wasrefluxed for 6 h. After being cooled to room temperature, the reactionmixture was poured into cold water (10 ml) and extracted with ethylacetate (15 ml). The organic layer was dried over MgSO₄, concentratedand chromatographed (dichloromethane) to yield 12b (0.162 g, 0.34 mmol,68%) as a white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.67-1.76 (m, 1H), 2.19-2.31 (m, 1H),2.59-2.74 (m, 4H), 7.40-7.44 (m, 3H), 7.47-7.53 (m, 2H), 7.64-7.67 (m,2H), 7.79-7.82 (m, 2H), 7.88 (dd, J=8.3 Hz, J₂=1.8 Hz, 1H), 7.97 (d,J=8.2 Hz, 1H), 8.02 (d, J=1.8 Hz, 1H); ¹³C NMR (CDCl₃, 100 MHz) δ 13.7,31.5, 67.5, 110.0, 114.9, 122.0 (q, J=272.6 Hz), 127.1 (q, J=4.7 Hz),127.3, 128.1, 128.7, 129.0, 130.2, 132.3, 133.5 (q, J=33.2 Hz), 134.2,135.2, 137.2, 139.6, 142.8, 174.9, 179.9.

Example 13 13-1). 1-(2-naphthylamino)cyclobutanecarbonitrile, 13a

Trimethylsilyl cyanide (0.27 ml, 2 mmol) was added dropwise to a mixtureof 2-aminonaphthalene (0.143 g, 1 mmol) and cyclobutanone (0.098 g, 1.4mmol). The reaction mixture was stirred at room temperature for 12 h andthen concentrated under vacuum to obtain a brown liquid which wassubjected to chromatography (dichloromethane) to yield 13a (0.209 g,0.94 mmol, 94%) as a yellow solid.

13-2).4-(8-oxo-6-thioxo-5-(4-biphenyl)-5,7-diazaspiro[3.4]oct-7-yl)-2-trifluoromethylbenzonitrile,12b, [RD85]

A mixture of 1a (0.137 g, 0.6 mmol) and 13a (0.111 g, 0.5 mmol) in dryDMF (0.2 ml) was stirred at room temperature for 3 days. To this mixturewere added methanol (5 ml) and HCl aq. (1 ml). The second mixture wasrefluxed for 6 h. After being cooled to room temperature, the reactionmixture was poured into cold water (10 ml) and extracted with ethylacetate (15 ml). The organic layer was dried over MgSO₄, concentratedand chromatographed (dichloromethane) to yield 12b (0.146 g, 0.325 mmol,65%) as a white powder.

¹H NMR (CDCl₃, 400 MHz) δ 158-1.68 (m, 1H), 2.17-2.29 (m, 1H), 2.61-2.75(m, 4H), 7.40 (dd, J=8.6 Hz, J₂=2.0 Hz, 1H), 7.58-7.65 (m, 2H),7.86-8.00 (m, 5H), 8.04 (J=1.8 Hz, 1H), 8.06 (d, J=8.6 Hz, 1H); ¹³C NMR(CDCl₃, 100 MHz) δ 13.7, 31.6, 67.7, 110.0, 114.9, 122.0 (q, J=272.6Hz), 126.8, 127.1 (q, J=4.8 Hz), 127.2, 127.7, 128.0, 128.3, 129.1,130.2, 132.2, 132.5, 133.4, 133.5 (q, J=33.1 Hz), 133.6, 135.2, 137.2,175.0, 180.1.

Example 14 14-1). 2-(4-methyl-2-pyridinamino)-2-methylpropanenitrile,14a

Trimethylsilyl cyanide (0.27 ml, 2 mmol) was added dropwise to a mixtureof 2-amino-4-methylpyridine (0.108 g, 1 mmol) and acetone (0.58 g, 10mmol). The reaction mixture was stirred at room temperature for 6 daysand then concentrated under vacuum to obtain a brown liquid which wassubjected to chromatography (dichloromethane: acetone, 60:40) to yield14a (0.133 g, 0.76 mmol, 76%) as a white solid.

14-2).4-[4,4-dimethyl-3-(4-methylpyridin-2-yl)-5-oxo-2-thioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile,14b, [RD83]

A mixture of 1a (0.91 g, 0.4 mmol) and 14a (0.053 g, 0.3 mmol) in dryDMF (0.2 ml) was stirred at room temperature for 6 days. To this mixturewere added methanol (5 ml) and HCl aq. (1 ml). The second mixture wasrefluxed for 5 h. After being cooled to room temperature, the reactionmixture was poured into cold water (10 ml) and extracted with ethylacetate (15 ml). The organic layer was dried over MgSO₄, concentratedand chromatographed (dichloromethane) to yield 14b (0.07 g, 0.174 mmol,58%) as a white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.70 (s, 6H), 2.44 (s, 3H), 7.19 (d, J=4.4 Hz,1H), 7.45 (t, J=0.6 Hz, 1H), 7.82 (dd, J₁=8.2 Hz, J₂=1.8 Hz, 1H), 7.95(d, J=1.8 Hz, 1H), 7.97 (d, J=8.2 Hz, 1H), 8.47 (d, J=5.0 Hz, 1H); ¹³CNMR (CDCl₃, 100 MHz) δ 21.1, 24.1, 67.1, 110.2, 114.8, 121.9 (q, J=272.6Hz), 124.4, 125.1, 127.3 (q, J=4.8 Hz), 132.4, 133.5 (q, J=33.2 Hz),135.3, 137.1, 149.2, 149.5, 150.0, 175.2, 179.0.

Example 15 15-1). 2-(2-pyridinamino)-2-methylpropanenitrile, 15a

Trimethylsilyl cyanide (0.27 ml, 2 mmol) was added dropwise to a mixtureof 2-aminopyridine (0.094 g, 1 mmol) and acetone (0.58 g, 10 mmol). Thereaction mixture was stirred at room temperature for 6 days and thenconcentrated under vacuum to obtain a brown liquid which was subjectedto chromatography (dichloromethane: acetone, 60:40) to yield 15a (0.131g, 0.81 mmol, 81%) as a white solid.

15-2).4-[4,4-dimethyl-3-(4-pyridin-2-yl)-5-oxo-2-thioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile,15b, [RD82]

A mixture of 1a (0.91 g, 0.4 mmol) and 15a (0.048 g, 0.3 mmol) in dryDMF (0.3 ml) was stirred at room temperature for 10 days. To thismixture were added methanol (5 ml) and of HCl aq. (1 ml). The secondmixture was refluxed for 5 h. After being cooled to room temperature,the reaction mixture was poured into cold water (10 ml) and extractedwith ethyl acetate (15 ml). The organic layer was dried over MgSO₄,concentrated and chromatographed (dichloromethane) to yield 15b (0.059g, 0.153 mmol, %) as a white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.73 (s, 6H), 7.38 (dd, J=7.3 Hz, J₂=5.4 Hz,1H), 7.71 (d, J=8.0 Hz, 1H), 7.87 (dd, J₁=8.2 Hz, J₂=1.8 Hz, 1H), 7.95(td, J=7.8 Hz, J₂=1.8 Hz, 1H), 7.95 (d, J=1.3 Hz, 1H), 7.98 (d, J=8.2Hz, 1H), 8.62 (dd, J=4.7 Hz, J₂=1.3 Hz, 1H); ¹³C NMR (CDCl₃, 100 MHz) δ24.2, 67.1, 110.3, 114.8, 121.9 (q, J=272.6 Hz), 123.7, 123.8, 127.3 (q,J=4.8 Hz), 132.4, 133.6 (q, J=33.2 Hz), 135.3, 137.1, 138.2, 149.5,149.6, 175.1, 179.0.

Example 16 16-1).1-(5-methyl-2H-pyrazol-3-ylamino)-cyclobutanecarbonitrile, 16a

Trimethylsilyl cyanide (0.532 ml, 4.0 mmol) was added dropwise to themixture of 3-amino-5-methylpyrazole (0.194 g, 2.0 mmol) andcyclobutanone (0.154 g, 2.2 mmol). The reaction mixture was stirred atroom temperature for 40 h and then concentrated under vacuum to obtain adark liquid which was subjected to chromatography (dichloromethane) toyield 16a (0.267 g, 1.52 mmol, 76%) as a off-white powder.

16-2).4-[5-(5-methyl-2H-pyrazol-3-yl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-7-yl]-2-trifluoromethyl-benzonitrile,16b, [RD84]

A mixture of 1a (0.0684 g, 0.3 mmol) and 16a (0.053 g, 0.3 mmol) in dryDMF (0.2 ml) was stirred at room temperature for 4 days. To this mixturewere added methanol (10 ml) and HCl aq. 2N (2 ml). The second mixturewas refluxed for 5 h. After being cooled to room temperature, thereaction mixture was poured into cold water (30 ml) and extracted withethyl acetate (30 ml). The organic layer was dried over MgSO₄,concentrated and chromatographed (dichloromethane:acetone, 97:3) toyield 16b (0.0826 g, 0.2 mmol, 67%) as a white powder.

¹H NMR (acetone d₆, 400 MHz) Δ Δ 1.66-1.76 (m, 1H), 2.00-2.07 (m, 1H),3.35 (s, 3H), 2.56-2.63 (m, 2H), 2.85-2.93 (m, 2H), 8.04 (dd, J=8.2 Hz,J₂=1.6 Hz, 1H), 8.18 (d, J=1.6 Hz, 1H), 8.22 (d, J=8.2 Hz, 1H), 11.99(s, 1H); ¹³C NMR (acetone d₆, 100 MHz) δ 10.2, 13.1, 31.1, 67.4, 102.5,109.1, 114.8, 122.5 (q, J=271.4 Hz), 127.8 (q, J=4.8 Hz), 131.9 (q,J=33.6 Hz), 133.6, 135.6, 138.4, 139.9, 145.0, 175.0, 179.6.

Example 174-[3-(4-hydroxyphenyl)-4,4-dimethyl-2,5-dithioxoazolidin-1-yl]-2-trifluoromethylbenzonitrile,17a, [RD59]

A mixture of 3c (0.081 g, 0.2 mmol) and Lawesson reagent (0.097 g, 0.24mmol) in toluene (3 ml) was heated to reflux for 15 h. After beingcooled to room temperature, the reaction mixture was poured into coldwater (10 ml) and extracted with ethyl acetate (10 ml). The organiclayer was dried over MgSO₄, concentrated and chromatographed(dichloromethane:pentane, 9:1) to yield 17a (0.0185 g, 0.044 mmol, 22%)as a white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.65 (s, 6H), 6.95-6.97 (m, 2H), 7.15-7.18 (m,2H), 7.75 (d, J=8.2 Hz, 1H), 7.86 (d, J=1.8 Hz, 1H), 7.98 (dd, J=8.2 Hz,J₂=1.8 Hz, 1H); ¹³C NMR (CDCl₃, 100 MHz) 27.9, 77.8, 110.9, 114.7,116.7, 121.9 (q, J=272.6 Hz), 128.1 (q, J=4.8 Hz), 129.1, 130.7, 133.3,133.5 (q, J=33.2 Hz), 135.5, 140.3, 156.8, 179.9, 207.9.

Example 184-[3-(4-hydroxyphenyl)-4,4-dimethyl-2,5-dioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile,18a, [RD60]

Hydrogen peroxide, 30% (3 ml, 26 mmol) was added dropwise to a solutionof 3c (0.121 g, 0.4 mmol) in glacial acetic acid (3 ml). The mixture wasstirred at room temperature for 12 h and then 20 ml of ethyl acetate wasadded. The organic layer was washed with water (3×15 ml), dried overmagnesium sulfate, concentrated and chromatographed (dichloromethane) toyield 18a (0.102 g, 0.261 mmol, 87%) as a white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.52 (s, 6H), 6.70-6.73 (m, 2H), 7.01-7.04 (m,2H), 7.92 (d, J=8.4 Hz, 1H), 8.00 (dd, J=8.4 Hz, J₂=1.8 Hz, 1H), 8.15(d, J=1.8 Hz, 1H); ¹³C NMR (CDCl₃, 100 MHz) δ 23.7, 63.7, 108.4, 115.0,116.7, 121.9 (q, J=272.6 Hz), 123.5 (q, J=4.8 Hz), 124.0, 128.5, 130.5,133.6 (q, J=33.2 Hz), 135.5, 136.2, 153.4, 157.2, 174.5.

Example 19 19-1).3-fluoro-2-methyl-2-(4-methylphenyl)aminopropionitrile, 19a

Trimethylsilyl cyanide (0.146 ml, 1.1 mmol) was added dropwise to themixture of p-toluidine (0.107 g, 1 mmol) and fluoroacetone (0.082 g, 1.1mmol). The reaction mixture was stirred at room temperature for 12 h andthen concentrated under vacuum to obtain a brown liquid which wassubjected to chromatography (dichloromethane) to yield 19a (0.179 g,0.93 mmol, 93%) as a yellowish solid.

19-2).4-(4-fluoromethyl-4-methyl-5-oxo-2-thioxo-3-(4-methylphenyl)imidazolidin-1-yl)-2-trifluoromethylbenzonitrile,19b, [RD68]

A mixture of 1a (0.16 g, 0.7 mmol) and 19a (0.096 g, 0.5 mmol) in dryDMF (0.3 ml) was stirred at room temperature for 48 h. To this mixturewere added methanol (10 ml) and HCl aq. 2N (2 ml). The second mixturewas refluxed for 6 h. After being cooled to room temperature; thereaction mixture was poured into cold water (30 ml) and extracted withethyl acetate (30 ml). The organic layer was dried over MgSO₄,concentrated and chromatographed (dichloromethane) to yield 19b (0.168g, 0.4 mmol, 80%) as a white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.49 (s, 3H), 2.44 (s, 3H), 4.35 (dd, J=47.2Hz, J₂=10.0 Hz, 1H), 4.71 (dd, J=45.2 Hz, J₂=10 Hz, 1H), 7.22-7.26 (m,2H), 7.35-7.39 (m, 2H), 7.82 (dd, J₁=8.2 Hz, J₂=1.8 Hz, 1H), 7.93 (d,J=1.8 Hz, 1H), 7.98 (d, J=8.2 Hz, 1H); ¹³C NMR (CDCl₃, 100 MHz) δ 17.0(d, J=4.6 Hz), 21.3, 69.3 (d, J=18.3 Hz), 81.9 (d, J=179.5 Hz), 109.9,114.8, 121.8 (q, J=272.6 Hz), 127.2 (q, J=4.7 Hz), 129.3, 130.9, 131.6,132.3, 133.3 (q, J=33.2 Hz), 135.3, 137.0, 140.5, 174.1, 181.4; ¹⁹F NMR(CDCl₃, 376 MHz) δ−62.5, 110.9.

Example 20 20-1).2-methyl-2-(4-trifluoromethylphenyl)aminopropanenitrile, 20a

A mixture of 4-trifluoromethylaniline (1.61 g, 10 mmol), acetonecyanohydrin (5 ml) and magnesium sulfate (2 g) was heated to 80° C. andstirred for 12 h. To the medium was added ethyl acetate (50 ml) and thenwashed with water (3×30 ml). The organic layer was dried over MgSO₄ andconcentrated under vacuum to dryness to yield 20a (2.166 g, 9.5 mmol,95%) as brown solid.

20-2).4-(4,4-dimethyl-5-oxo-2-thioxo-3-(4-trifluoromethylphenyl)imidazolidin-1-yl)-2-trifluoromethylbenzonitrile,20b, [RD66]

A mixture of 1a (0.114 g, 0.5 mmol) and 20a (0.092 g, 0.4 mmol) in dryDMF (0.3 ml) was stirred at room temperature for 48 h. To this mixturewere added methanol (10 ml) and HCl aq. (3 ml). The second mixture wasrefluxed for 6 h. After being cooled to room temperature, the reactionmixture was poured into cold water (20 ml) and extracted with ethylacetate (20 ml). The organic layer was dried over MgSO₄, concentratedand chromatographed (dichloromethane) to yield 20b (0.117 g, 0.256 mmol,64%) as a white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.61 (s, 6H), 7.45-7.49 (m, 2H), 7.80-7.83 (m,2H), 7.85 (dd, J=8.3 Hz, J₂=1.8 Hz, 1H), 7.97 (d, J=1.8 Hz, 1H), 7.99(d, J=8.2 Hz, 1H); ¹³C NMR (CDCl₃, 100 MHz) δ 23.8, 66.6, 110.3, 114.8,121.8 (q, J=272.6 Hz), 123.5 (q, J=271.1 Hz), 127.0 (q, J=4.6 Hz), 127.1(q, J=4.7 Hz), 130.3, 131.9 (q, J=32.9 Hz), 132.2, 133.5 (q, J=33.3 Hz),135.3, 136.9, 138.4, 174.6, 179.9.

Example 21 21-1).3-chloro-2-chloromethyl-2-(4-methylphenyl)aminopropanenitrile, 21a

Trimethylsilyl cyanide (0.27 ml, 2 mmol) was added dropwise to a mixtureof p-toluidine (0.107 g, 1 mmol) and 1,3-dichloroacetone (0.254 g, 2mmol). The reaction mixture was heat to 80° C. and stirred for 6 h. Tothe mixture was added 20 ml of ethyl acetate and then wash with water(2×20 ml). The organic layer was dried over MgSO₄, concentrated andchromatographed (dichloromethane) to yield 21a (0.192 g, 0.79 mmol, 79%)as a brown powder.

21-2).4-(4,4-bischloromethyl-5-oxo-2-thioxo-3-(4-methylphenyl)imidazolidin-1-yl)-2-trifluoromethylbenzonitrile,21b, [RD67]

A mixture of 1a (0.16 g, 0.7 mmol) and 21a (0.122 g, 0.5 mmol) in dryDMF (0.5 ml) was stirred at room temperature for 10 days. To thismixture were added methanol (10 ml) and of HCl aq. 2N (2 ml). The secondmixture was refluxed for 6 h. After being cooled to room temperature,the reaction mixture was poured into cold water (20 ml) and extractedwith ethyl acetate (30 ml). The organic layer was dried over MgSO₄,concentrated and chromatographed (dichloromethane) to yield 21b (0.09 g,0.19 mmol, 38%) as a white powder.

¹H NMR (CDCl₃, 400 MHz) δ 2.44 (s, 3H), 3.54 (d, J=11.8 Hz, 2H), 3.93(d, J=11.8 Hz, 2H), 7.37-7.40 (m, 2H), 7.48-7.51 (m, 2H), 7.79 (dd,J₁=8.2 Hz, J₂=1.8 Hz, 1H), 7.88 (d, J=1.8 Hz, 1H), 7.98 (d, J=8.2 Hz,1H); ¹³C NMR (CDCl₃, 100 MHz) δ 21.4, 42.8, 74.3, 110.7, 114.7, 121.7(q, J=272.6 Hz), 127.2 (q, J=4.7 Hz), 128.8, 131.0, 131.1, 132.4, 133.8(q, J=33.2 Hz), 135.5, 136.9, 140.9, 169.5, 182.5.

Example 22 22-1). 1-(4-methylphenyl)aminocyclohexanenitrile, 22a

Sodium cyanide (0.245 g, 5 mmol) was added to a mixture of anthranilicacid (0.411 g, 3 mmol) and acetone (1 ml, 13.6 mmol) in acetic acid 90%(3 ml). The reaction mixture was stirred at room temperature for 12 hand then 50 ml of ethyl acetate was added. The organic layer was washedwith brine (3×30 ml). The organic layer was dried over magnesiumsulfate, concentrated and chromatographed (dichloromethane:acetone,90:10) to yield 22a (0.551 g, 2.7 mmol, 90%) as a brown solid.

22-2).2-[3-(4-cyano-3-trifluoromethylphenyl)-5,5-dimethyl-4-oxo-2-thioxoimidazolidin-1-yl]benzoicacid, 22b, [RD65]

A mixture of 1a (0.114 g, 0. mmol) and 22a (0.103 g, 0.5 mmol) in dryDMF (0.5 ml) was stirred at room temperature for 3 days. To this mixturewere added methanol (10 ml) and HCl aq. 2N, (3 ml). The second mixturewas refluxed for 6 h. After being cooled to room temperature, thereaction mixture was poured into cold water (20 ml) and extracted withethyl acetate (30 ml). The organic layer was dried over MgSO₄,concentrated and chromatographed (ethyl acetate:pentane, 2:1) to yield22b (0.143 g, 0.33 mmol, 66%) as a white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.47 (s, 3H), 1.78 (s, 3H), 7.39 (d, J=7.7 Hz,1H), 7.63 (t, J=7.7 Hz, 1H) 7.76-7.82 (m, 2H), 7.90-7.98 (m, 2H), 8.22(d, J=6.8 Hz, 1H), 8.96 (bs, 1H); ¹³C NMR (CDCl₃, 100 MHz) δ 20.6, 26.2,67.6, 110.1, 114.8, 121.9 (q, J=272.6 Hz), 127.2 (q, J=4.7 Hz), 128.9,131.0, 130.2, 132.5, 133.2 (q, J=33.3 Hz), 133.7, 134.7, 135.4, 135.8,137.3, 169.8, 175.3, 180.7.

Example 23 23-1). 1-(2-methylphenyl)aminocyclobutanenitrile, 23a

Trimethylsilyl cyanide (0.66 ml, 5 mmol) was added dropwise to themixture of p-toluidine (0.321 g, 3 mmol) and cyclobutanone (0.28 g, 4mmol). The reaction mixture was stirred at room temperature for 6 h andthen concentrated under vacuum to obtain a brown liquid which wassubjected to chromatography (dichloromethane) to yield 23a (0.541 g,2.91 mmol, 97%) as a yellowish solid.

23-2).4-(8-oxo-6-thioxo-5-(2-methylphenyl)-5,7-diazaspiro[3.4]oct-7-yl)-2-trifluoromethylbenzonitrile,23b, [RD71]

A mixture of 1a (0.114 g, 0.5 mmol) and 23a (0.093 g, 0.5 mmol) in dryDMF (0.3 ml) was stirred at room temperature for 3 days. To this mixturewere added methanol (10 ml) and HCl aq. 2N, (3 ml). The second mixturewas refluxed for 6 h. After being cooled to room temperature, thereaction mixture was poured into cold water (20 ml) and extracted withethyl acetate (30 ml). The organic layer was dried over MgSO₄,concentrated and chromatographed (dichloromethane) to yield 23b (0.116g, 0.28 mmol, 56%) as a white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.63-1.69 (m, 1H), 2.26 (s, 3H), 2.28-2.41 (m,2H), 2.58-2.76 (m, 3H), 7.21 (d, J=7.6 Hz, 1H), 7.39-7.49 (m, 3H), 7.89(dd, J=8.2 Hz, J₂=1.8 Hz, 1H), 7.97 (d, J=8.2 Hz, 1H), 8.00 (d, J=1.8Hz, 1H); ¹³C NMR (CDCl₃, 100 MHz) δ 14.2, 18.0, 30.7, 32.2, 67.6, 109.9,114.9, 121.9 (q, J=272.6 Hz), 127.0 (q, J=4.7 Hz), 127.5, 129.8, 130.2,131.9, 132.3, 133.4, 133.5 (q, J=34.3 Hz), 135.2, 135.8, 137.1, 138.0,175.3, 178.7.

Example 24 24-1). 1-aminocyclopentanecarbonitrile, 24a

Ammonia anhydrous was bubble into a mixture of cyclopentanone (0.452 g)and trimethylsilyl cyanide (0.66 ml, 5 mmol). The excess of ammonia wasrefluxed by a dry ice-acetone condenser. After 1 h of reflux, theammonia was allowed to degas form the medium and then the remainingmixture was concentrated under vacuum to yield 24a (0.522 g, 4.75 mmol,95%) as a colorless liquid.

24-2).4-(4-imino-2-thioxo-1,3-diazaspiro[4.4]non-3-yl)-2-trifluoromethylbenzonitrile,24b

Triethylamine (0.101 g, 0.1 mmol) was added to a solution of 1a (0.684g, 3 mmol) and 24a (0.33 g, 3 mmol) in dry THF (5 ml). The reactionmixture was stirred at room temperature for 5 h and then concentrated toyield a brown residue which was subjected to flash chromatography(dichloromethane/acetone, 93:7) to afford 24b (0.741 g, 2.19 mmol, 73%).

24-3).4-(4-oxo-2-thioxo-1,3-diazaspiro[4.4]non-3-yl)-2-trifluoromethylbenzonitrile,24c, [RD77]

A mixture of 24b (0.741 g, 2.19 mmol) in HCl aq., 2N (4 ml) and methanol(20 ml) was heated to reflux for 1 h. After being cooled to roomtemperature, the reaction mixture was poured into cold water (20 ml) andextracted with ethyl acetate (40 ml). The organic layer was dried overMgSO₄, concentrated and chromatographed (dichloromethane) to yield 24c(0.72 g, 2.12 mmol, 97%) as a white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.86-190 (m, 2H), 1.96-2.05 (m, 4H), 2.26-2.30(m, 2H), 7.80 (dd, J₁=8.2 Hz, J₂=1.8 Hz, 1H), 7.92 (d, J=1.8 Hz, 1H),7.97 (d, J=8.2 Hz, 1H) 8.20 (bs, NH); ¹³C NMR (CDCl₃, 100 MHz) δ 25.3,38.1, 71.0, 110.1, 114.8, 121.8 (q, J=272.7 Hz), 126.8 (q, J=4.7 Hz),131.9, 133.6 (q, J=34.3 Hz), 135.3, 136.7, 176.1, 179.8.

Example 25 25).4-[1-(4-nitrophenyl)-4-oxo-2-thioxo-1,3-diazaspiro[4.4]non-3-yl]-2-trifluoromethylbenzonitrile,25a, [RD55]

A mixture of 25c (0.0678 g, 0.2 mmol),1,8-Diazabicyclo[5.4.0]undec-7-ene (0.05 g, 0.33 mmol) and4-fluoronitrobenzene (0.056 g, 0.4 mmol) in dimethylformamide (0.5 ml)was placed under argon in a sealed-tube and heated to 130° C. for 40 h.The reaction mixture was poured into ethyl acetate (5 ml) and washedwith water (2×10 ml). The organic layer was dried over MgSO₄,concentrated and chromatographed (dichloromethane) to yield 25a (0.038g, 0.084 mmol, 42%) as a white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.53-1.56 (m, 2H), 1.90-1.93 (m, 2H),2.14-2.18 (m, 2H), 2.37-2.40 (ni, 2H), 7.54-7.57 (m, 2H), 7.85 (dd,J=8.2 Hz, J₂=1.8 Hz, 7.97 (d, J=1.8 Hz, 1H), 7.98 (d, J=8.2 Hz, 1H),8.39-8.43 (m, 2H); ¹³C NMR (CDCl₃, 100 MHz) δ 25.2, 36.5, 75.3, 110.3,114.8, 121.8 (q, J=272.6 Hz), 125.2, 127.0 (q, J=4.7 Hz), 131.4, 132.1,133.6 (q, J=34.3 Hz), 135.3, 136.9, 141.7, 148.1, 175.6, 180.2.

Example 26 26).4-[1-(4-cyanophenyl)-4-oxo-2-thioxo-1,3-diazaspiro[4.4]non-3-yl]-2-trifluoromethylbenzonitrile,26a, [RD54]

A mixture of 24c (0.0678 g, 0.2 mmol),1,8-diazabicyclo[5.4.0]undec-7-ene (0.061 g, 0.4 mmol) and4-fluorocyanobenzene (0.048 g, 0.4 mmol) in dimethylformamide (0.5 ml)was placed under argon in a sealed-tube and heated to 140° C. for 5days. The reaction mixture was poured into ethyl acetate (5 ml) andwashed with water (2×10 ml). The organic layer was dried over MgSO₄,concentrated and chromatographed (dichloromethane) to yield 26a (0.023g, 0.052 mmol, 26%) as a white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.51-1.55 (m, 2H), 1.90-1.93 (m, 2H),2.12-2.16 (m, 2H), 2.33-2.38 (m, 2H), 7.47-7.50 (m, 2H), 7.81-7.87 (m,3H), 7.95-7.99 (m, 2H); ¹³C NMR (CDCl₃, 100 MHz) δ 25.2, 36.5, 75.3,110.3, 113.9, 114.7, 117.5, 121.8 (q, J=272.6 Hz), 127.0 (q, J=4.8 Hz),131.2, 132.1, 133.6 (q, J=34.3 Hz), 133.8, 135.3, 136.9, 140.0, 175.6,180.1.

Example 27 27-1).1-methyl-4-(4-methylpheaylamino)piperidine-4-carbonitrile, 27a

Sodium cyanide (0.318 g, 6.5 mmol) was added to a mixture of p-toluidine(0.535 g, 5 mmol) and 1-methyl-4-piperidinone (0.678 g, 6 mmol) inacetic acid 90% (5 ml). The reaction mixture was stirred at roomtemperature for 6 h and then 100 ml of dichloromethane was added. Theorganic layer was washed with a solution NaOH, 2N (2×50 ml), dried overmagnesium sulfate, concentrated and chromatographed (DCM and thenacetone) to obtained 27a (0.722 g, 3.15 mmol, 63%).

27.2).4-(4-imino-8-methyl-2-thioxo-1-(4-methylphenyl)-1,3,8-triazaspiro[4.5]dec-3-yl)-2-trifluoromethylbenzonitrile,27b

Triethylamine (0.02, 0.2 mmol) was added to a solution of 1a (0.228 g, 1mmol) and 27a (0.114 g, 0.5 mmol) in dry THF (2 ml). The reactionmixture was stirred at room temperature for 20 h and then concentratedto yield a dark residue which was subjected to flash chromatography(dichloromethane/acetone, 90:10, and then acetone) to afford 27b (0.059g, 0.13 mmol, 26%).

27-3). 4-(8-methyl-4-oxo-2-thioxo-1-(4-methylphenyl)-1,3,8-triazaspiro[4.5]dec-3-yl)-2-trifluoromethylbenzonitrile,27c, [RD53]

A mixture of 27b (0.059 g, 0.13 mmol) in HCl aq., 2N (1 ml) and methanol(3 ml) was heated to reflux for 2 h. After being cooled to roomtemperature, the reaction mixture was poured into cold water (5 ml) andextracted with ethyl acetate (10 ml). The organic layer was dried overMgSO₄, concentrated and chromatographed (dichloromethane:acetone, 60:40)to yield 27c (0.055 g, 0.012 mmol, 92%) as a white powder.

¹H NMR (Acetone-d₆, 400 MHz) δ 1.93-1.99 (m, 1H), 2.00-2.04 (m, 1H),2.18 (s, 3H), 2.24-2.28 (m, 2H), 2.38 (s, 3H), 2.61-2.72 (m, 4H),7.18-7.20 (m, 2H), 7.32-7.35 (m, 2H), 8.03 (dd, J₁=8.2 Hz, J₂=1.8 Hz,1H), 8.16 (d, J=1.8 Hz, 1H), 8.22 (d, J=8.2 Hz, 1H); ¹³C NMR(Acetone-d₆, 100 MHz) δ 20.3, 31.4, 45.1, 49.8, 65.1, 109.1, 114.8,122.4 (q, J=275.1 Hz), 127.7 (q, J=4.8 Hz), 130.0, 130.5, 131.9 (q,J=32.6 Hz), 132.6, 133.5, 135.6, 138.3, 139.4, 174.0, 180.6.

Example 284-(8-methyl-4-oxo-2-thioxo-1,3,8-triazaspiro[4.5]dec-3-yl)-2-trifluoromethylbenzonitrile,28a, [RD52]

Compound 28a was synthesized according to the procedure described inU.S. Pat. No. 5,958,936.

¹H NMR (Acetone-d₆, 400 MHz) δ 1.93-2.00 (m, 2H), 2.09-2.16 (m, 2H),2.25 (s, 3H), 2.42-2.49 (m, 2H), 2.75-2.80 (m, 2H), 7.97 (dd, J=8.2 Hz,J₂=1.8 Hz, 1H), 8.11 (d, J=1.8 Hz, 1H), 8.20 (d, J=8.2 Hz, 1H), 9.80(bs, NH); ¹³C NMR (Acetone-d₆, 100 MHz) δ 32.9, 45.4, 50.1, 62.3, 109.1,114.8, 122.4 (q, J=271.6 Hz), 127.5 (q, J=4.8 Hz), 131.8 (q, J=32.7 Hz),133.2, 135.6, 135.6, 138.0, 175.2, 180.4.

Example 294-[3-(4-hydroxybutyl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile,RU 59063

Compound RU 59063 was synthesized according to the procedure describedby Teutsch et al [J. Steroid. Biochem. Molec. Biol. 1994, 48(1),111-119].

¹H NMR (CDCl₃, 400 MHz) δ 1.55 (s, 6H), 1.58-1.62 (m, 2H), 1.86-1.89 (m,2H), 2.25 (bs, OH), 3.65-3.71 (m, 4H), 7.74 (dd, J=8.0 Hz, J₂=1.8 Hz,1H), 7.92 (d, J=1.8 Hz, 1H), 7.98 (d, J=8.0 Hz, ¹H); ¹³C NMR (CDCl₃, 100MHz) δ 23.1, 24.7, 29.6, 43.9, 61.7, 65.2, 109.7, 114.9, 121.9 (q,J=272.6 Hz), 127.1 (q, J=4.8 Hz), 132.2, 133.7 (q, J=34.3 Hz), 135.2,137.2, 175.3, 178.2.

Example 30 30-1). 1-methylaminocyclobutanecarbonitrile, 30a

Methylamine was bubbled into a refrigerated mixture of cyclobutanone(0.21 g, 3 mmol) and trimethylsilyl cyanide (0.396 g, 4 mmol) until thevolume doubled. The mixture was stirred 3 h and then concentrated todryness to obtain 30a (0.33 g, quantitative).

30-2).4-(5-methyl-8-oxo-6-thioxo-5,7-diazaspiro[3.4]oct-7-yl)-2-trifluoromethylenzonitrile,30b, [RD73]

A mixture of 1a (0.114 g, 0.5 mmol) and 30a (0.055 g, 0.5 mmol) in dryDMF (0.2 ml) was stirred at room temperature for 0.5 h. To this mixturewere added 10 ml of methanol and 2 ml of 2N HCl. The second mixture wasrefluxed for 2 h. After being cooled to room temperature, the reactionmixture was poured into cold water (20 ml) and extracted with ethylacetate (30 ml). The organic layer was dried over MgSO₄, concentratedand chromatographed (dichloromethane) to yield 30b (0.148 g, 0.435 mmol,87%) as a white powder.

¹H NMR (CDCl₃, 400 MHz) δ1.95-2.06 (m, 1H), 2.21-2.32 (m, 1H), 2.58-2.71(m, 4H), 3.44 (s, 3H), 7.77 (dd, J=8.2 Hz, J₂=2.0 Hz, 1H), 7.89 (d,J=2.0 Hz, 1H), 7.93 (d, J=8.2 Hz, 1H); ¹³C NMR (CDCl₃, 100 MHz) δ 13.7,30.3, 30.4, 66.1, 109.7, 114.9, 121.9 (q, J=272.6 Hz), 126.9 (q, J=4.8Hz), 132.1, 133.2 (q, J=34.3 Hz), 135.2, 137.3, 175.1, 178.7.

30-3).4-(5-methyl-6,8-dioxo-5,7-diazaspiro[3.4]oct-7-yl)-2-trifluoromethylbenzonitrile,30c, [RD74]

Hydrogen peroxide (2 ml, 30%) was added to the mixture of 30b (0.068 g,0.2 mmol) in glacial acetic acid (3 ml). After being stirred at roomtemperature for 10 h, the reaction mixture was poured into ethyl acetate(20 ml) and then washed with water (2×20 ml). The organic layer wasdried over MgSO₄, concentrated and chromatographed(dichloromethane:acetone) to yield 30c (0.057 g, 0.176 mmol, 88%) as awhite powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.91-2.35 (m, 1H), 2.21-2.31 (m, 1H),2.50-2.61 (m, 4H), 3.12 (s, 3H), 7.89 (d, J=8.2 Hz, 1H), 7.97 (dd, J=8.2Hz, J₂=2.0 Hz, 1H), 8.12 (d, J=2.0 Hz, 1H), ¹³C NMR (CDCl₃, 100 MHz) δ13.9, 25.4, 29.3, 63.4, 108.1, 115.1, 121.6 (q, J=272.6 Hz), 122.9 (q,J=4.8 Hz), 127.9, 133.5 (q, J=34.3 Hz), 135.3, 136.5, 152.7, 174.4.

Example 31 31-1). 1-methylaminocyclopentanecarbonitrile, 31a

Methylamine was bubbled into a refrigerated mixture of cyclopentanone(0.252 g, 3 mmol) and trimethylsilyl cyanide (0.396 g, 4 mmol) until thevolume doubled. The mixture was stirred 3 h and then concentrated todryness to obtain 31a (0.372 g, quantitative).

31-2).4-(1-methyl-4-oxo-2-thioxo-1,3-diazaspiro[4.4]non-3-yl)-2-trifluoromethylbenzonitrile,31b, [RD75]

A mixture of 1a (0.114 g, 0.5 mmol) and 31a (0.062 g, 0.5 mmol) in dryDMF (0.2 ml) was stirred at room temperature for 0.5 h. To this mixturewere added 10 ml of methanol and 2 ml of 2N HCl. The second mixture wasrefluxed for 2 h. After being cooled to room temperature, the reactionmixture was poured into cold water (20 ml) and extracted with ethylacetate (30 ml). The organic layer was dried over MgSO₄, concentratedand chromatographed (dichloromethane) to yield 31b (0.159 g, 0.45 mmol,90%) as a white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.91-2.05 (m, 6H), 2.16-2.21 (m, 2H), 3.27 (s,3H), 7.77 (dd, J=8.2 Hz, J₂=1.8 Hz, 1H), 7.89 (d, J=1.8 Hz, 1H), 7.91(d, J=8.2 Hz, 1H); ¹³C NMR (CDCl₃, 100 MHz) δ 26.4, 30.3, 35.4, 73.2,109.5, 114.9, 121.9 (q, J=272.6 Hz), 126.9 (q, J=4.8 Hz), 132.2, 133.2(q, J=34.3 Hz), 135.2, 137.5, 176.8, 178.5.

31-3).4-(1-methyl-2,4-dioxo-1,3-diaza-spiro[4.4]non-3-yl)-2-trifluoromethylbenzonitrile,31c, [RD76]

Hydrogen peroxide (2 ml, 30%) was added to the mixture of 31b (0.07 g,0.2 mmol) in glacial acetic acid (3 ml). After being stirred at roomtemperature for 10 h, the reaction mixture was poured into ethyl acetate(20 ml) and then washed with water (2×20 ml). The organic layer wasdried over MgSO₄, concentrated and chromatographed(dichloromethane:acetone) to yield 31c (0.057 g, 0.168 mmol, 84%) as awhite powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.88-1.99 (m, 6H), 2.12-2.17 (m, 2H), 2.98 (s,3H), 7.88 (d, J=8.2 Hz, 1H), 7.97 (dd, J=8.2 Hz, J₂=1.8 Hz, 1H), 8.12(d, J=1.8 Hz, 1H); ¹³C NMR (CDCl₃, 100 MHz) δ 25.2, 26.5, 34.8, 70.1,108.0, 115.1, 122.0 (q, J=272.5 Hz), 122.9 (q, J=4.9 Hz), 127.9, 133.5(q, J=32.9 Hz), 135.3, 136.6, 152.7, 176.1.

Example 324-(8-methylimino-6-thioxo-5-p-tolyl-5,7-diaza-spiro[3.4]oct-7-yl)-2-trifluoromethyl-benzonitrile,32a, [RD90]

A mixture of 7b (0.042 g, 0.1 mmol), DBU (0.023 g, 0.15 mmol) andiodomethane (0.073 g, 0.5 mmol) in DMF (0.3 ml) was stirred for 15 h atroom temperature. After DMF being evaporated, the medium waschromatographed (dichloromethane) to yield 32a (0.011 g, 0.026 mmol,26%) as white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.58-1.65 (m, 1H), 2.04-2.13 (m, 1H), 2.45 (s,3H), 2.70-2.77 (m, 2H), 3.06-3.10 (m, 2H), 3.58 (s, CH₃—N, major isomer)[2.70 (s, CH₃—N, minor isomer)], 7.20-7.34 (m, 4H), 7.75-7.91 (m, 3H);(CDCl₃, 100 MHz) δ 12.6, 21.4, 30.2, 33.7 (35.3 for the other isomer),66.9, 109.1, 115.2, 122.1 (q, J=272.5 Hz), 128.5 (q, J=4.9 Hz), 129.8,130.4, 130.6, 132.8, 133.2 (q, J=32.9 Hz), 133.5, 134.9, 139.8, 157.0,180.2.

Example 33 1-[3-(4-cyano-3-trifluoromethyl-phenyl)-5,5-dimethyl-2-thioxo-1-p-tolyl-imidazolidin-4-ylidene]-3-ethyl-thiourea, 33a,[RD91]

A mixture of 5b (0.06 g, 0.149 mmol), ethylthioisocyanate (0.087 g, 1mmol) and CuI (0.01 g, 0.05 mmol) in DMF (0.1 ml) was heated undermicrowave for 45 minutes. Then the medium was washed with brine andextracted with ethyl acetate. The organic layer was dried over MgSO₄,concentrated and chromatographed (HPLC, alumina column) to yield 33a(0.054 g, 0.108 mmol, 72%) as white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.15 (t, J=7.23 Hz, 3H), 1.70 [1.75 minorisomer] (s, 6H), 2.42 (s, 3H), 3.28-3.39 (m, 21-1) [3.15-3.22 (m, 2H),minor isomer], 6.50 (bs, 1H) [6.93 (bs, 1H), minor isomer], 7.14-7.18(m, 2H), 7.32-7.35 (m, 2H), 7.77-7.94 (m, 3H); ¹³C NMR (CDCl₃, 100 MHz)δ 13.31 (13.83 minor), 21.3, 25.22 (24.89 minor), 40.31 (40.67 minor),68.1, 109.9, 114.9, 122.3 (q, J=272.5 Hz), 127.6 (q, J=4.9 Hz), 129.1,129.59 (129.55 minor), 130.52 (130.57 minor), 132.27 (132.15 minor),132.9 (q, J=32.9 Hz), 134.27 (134.15 minor), 134.9, 135.2, 156.33(156.06 minor), 180.28 (180.06 minor), 187.24 (186.63 minor).

Example 341-[7-(4-cyano-3-trifluoromethyl-phenyl)-6-thioxo-5-p-tolyl-5,7-diaza-spiro[3.4]oct-8-ylidine]-3-phenyl-thiourea,34a, [RD92]

A mixture of 7b (0.021 g, 0.05 mmol) and phenylthioisocyante (0.027 g,0.2 mmol) in DMF (0.3 ml) was stirred for 2 days at 60° C. After DMFbeing evaporated, the medium was chromatographed (dichloromethane) toyield 34a (0.015 g, 0.028 mmol, 57%) as white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.59-1.67 (m, 1H), 2.12-2.22 (m, 1H), 2.45 (s,3H), 2.61-2.71 (m, 2H), 2.81-2.87 (m, 2H), 7.18-7.27 (m, 6H), 7.33-7.41(m, 5H), 7.60-7.62 (m, 1H), 8.40 (bs, 1H); ¹³C NMR (CDCl₃, 100 MHz) δ13.6, 21.4, 32.3, 69.6, 110.7, 114.8, 121.6, 122.0 (q, J=272.5 Hz),126.3, 128.0 (q, J=4.9 Hz), 128.9, 129.4, 130.7, 132.5, 133.2 (q, J=32.9Hz), 134.1, 134.9, 137.7, 139.2, 140.2, 154.8, 180.3, 185.5.

Example 351-(4-Cyano-3-trifluoromethyl-phenyl)-3-[7-(4-cyano-3-trifluoromethyl-phenyl)-6-thioxo-5-p-tolyl-5,7-diaza-spiro[3.4]oct-8-ylidene]-thiourea,35a, [RD93]

A mixture of 1a (0.5.02 g, 2.2 mmol) and 7a (0.186 g, 1 mmol) in DMF (1ml) was stirred at room temperature. After 20 hours of stirring, themixture was concentrated under reduced pressure to yield an orangeviscous liquid, which was chromatographed (dichloromethane:acetone,99:1) to yield 35a (0.269 g, 0.42 mmol, 42%) as a yellow powder.

X-ray structure of 35a Example 36 36-1).1-(4-hydroxymethylphenylamino)-cyclobutanecarbonitrile, 36a

Trimethylsilyl cyanide (0.66 ml, 5 mmol) was added dropwise to a mixtureof 4-aminobenzoic acid (0.492 g, 4 mmol) and cyclobutanone (0.35 g, 5mmol) in dichloromethane (10 ml). The reaction mixture was stirred atroom temperature for 6 h and then concentrated under vacuum to obtain abrown liquid which was subjected to chromatography (dichloromethane) toyield 36a (0.677 g, 3.36 mmol, 84%) as a brown solid.

36-2).4-[8-(4-hydroxymethylphenyl)-5-oxo-7-thioxo-6-azaspiro[3.4]oct-6-yl]-2-trifluoromethyl-benzonitrile,36b, [RD110]

A mixture of 1a (0.342 g, 1.5 mmol) and 36a (0.21 g, 1 mmol) DMF (0.5ml) was stirred at room temperature for 24 h. To this mixture were addedmethanol (20 ml) and HCl aq. 2N (5 ml). The second mixture was refluxedfor 6 h. After being cooled to room temperature, the reaction mixturewas poured into cold water (40 ml) and extracted with ethyl acetate (60ml). The organic layer was dried over MgSO₄, concentrated andchromatographed (dichloromethane:acetone, 90:10) to yield 36b (0.296 g,0.69 mmol, 69%) as a white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.63-1.68 (m, 1H), 2.17-2.26 (m, 1H),2.52-2.68 (m, 4H), 4.75 (s, 2H), 7.30 (d, J=8.1 Hz, 2H), 7.58 (d, J=8.1Hz, 2H), 7.88 (dd, J=8.3 Hz, J₂=1.8 Hz, 1H), 7.95-7.98 (m, 2H); ¹³C NMR(CDCl₃, 100 MHz) δ 13.7, 31.5, 64.4, 67.5, 109.9, 114.9, 121.9 (q,J=272.6 Hz), 127.1 (q, J=4.7 Hz), 128.3, 130.0, 132.2, 133.3, 133.4 (q,J=33.2 Hz), 134.2, 137.2, 142.9, 174.9, 179.9.

Example 374-[5-(4-formylphenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]oct-7-yl]-2-trifluoromethyl-benzonitrile,37a, [RD114]

To a mixture of 36b (0.303 g, 0.7 mmol) and Dess-Martin periodinane(0.417 g, 1 mmol) in dichloromethane (5 ml) was added pyridine (1.01 g,1 mmol). The mixture was stirred for 2 hours at room temperature andthen ethyl ether (10 ml) was added to precipitate the by-product of thereaction. After filtration and concentration under reduced pressure, themixture was chromatographed (dichloromethane:acetone, 95:5) to yield 37a(0.24 g, 0.56 mmol, 80%) as white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.62-1.73 (m, 1H), 2.24-2.30 (m, 1H),2.50-2.58 (m, 2H), 2.69-2.75 (m, 2H), 7.53 (d, J=8.1 Hz, 2H), 7.85 (dd,J=8.3 Hz, J₂=1.8 Hz, 1H), 7.97-7.99 (m, 2H), 8.11 (d, J=8.1 Hz, 2H),10.12 (s, 1H); ¹³C NMR (CDCl₃, 100 MHz) δ 13.7, 31.7, 67.5, 110.2,114.8, 121.9 (q, J1=272.6 Hz), 127.0 (q, J=4.7 Hz), 129.1, 131.0, 131.2,132.2, 133.3 (q, J=33.2 Hz), 135.3, 136.9, 140.5, 174.5, 179.8, 190.8.

Example 384-{5-[4-(1-hydroxyethyl)-phenyl]-8-oxo-6-thioxo-5,7-diazaspiro[3.4]oct-7-yl}-2-trifluoromethyl-benzonitrile,38a [RD116]

The mixture of 37a (0.043 g, 0.1 mmol) and dry THF (1 ml) in aflamed-dried flash was placed under argon and cooled to −78° C. Then,methylmagnesium iodide (1.1 ml, 0.1 M) was added. The mixture wasstirred at −78° C. for 30 minutes and warmed slowly to room temperature.The medium was washed with water (3 ml) and extracted with ethyl acetate(10 ml). The organic layer was dried over MgSO₄, concentrated andchromatographed (dichloromethane:acetone, 95:5) to yield 38a (0.037 g,0.032 nmol. 82%) as a white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.57 (d, J=6.5 Hz, 3H), 1.61-1.71 (m, 1H),2.09 (d, J=3.2 Hz, OH), 2.16-2.28 (m, 1H), 2.52-2.60 (m, 2H), 2.63-2.69(m, 2H), 5.00 (dd, J_(j)=6.5 Hz, q, J₂=3.1 Hz, 1H), 7.29 (d, J=8.3 Hz,2H), 7.60 (d, J=8.2 Hz, 2H), 7.85 (dd, J=8.3 Hz, J₂=1.8 Hz, 1H),7.95-7.98 (m, 2H); ¹³C NMR (CDCl₃, 100 MHz) δ 13.7, 25.3, 31.5, 67.4,69.8, 110.0, 114.9, 121.9 (q, J=272.6 Hz), 127.0 (q, J=4.7 Hz), 127.1,129.9, 132.2, 133.4 (q, J=33.2 Hz), 134.1, 135.2, 137.1, 147.6, 174.9,179.9.

Example 393-{4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]oct-5-yl]-phenyl}-acrylicacid ethyl ester, 39a [RD117]

A mixture of 37a (0.043 g, 0.1 mmol) and(carbethoxyethylidene)triphenylphosphorane (0.039 g, 0.12 mmol) indichloromethane (2 ml) was stirred at room temperature for 10 hours. Themedium was concentrated and chromatographed (dichloromethane) to yield39a (0.048 g, 0.096 mmol, 96%) as white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.35 (t, J=7.1 Hz, 3H), 1.66-1.70 (m, 1H),2.19-2.65 (m, 1H), 2.51-2.69 (m, 2H), 2.66-2.72 (m, 2H), 4.28 (q, J=7.1Hz, 2H), 6.51 (d. J=16.1 Hz, 1H), 7.35 (d, J=8.3 Hz 2H), 7.72 (d, J=8.3Hz, 2H), 7.73 (d, J=16.1 Hz, 1H), 7.85 (dd, J₁=8.3 Hz, J₂=1.8 Hz, 1H),7.96-7.98 (m, 2H); ¹³C NMR (CDCl₃, 100 MHz) δ 13.7, 14.3, 31.6, 60.8,67.5, 110.0, 114.9, 120.5, 121.8 (q, J=272.6 Hz), 127.0 (q, J=4.7 Hz),129.5, 130.5, 132.2, 133.4 (q, J=33.2 Hz), 135.2, 136.0, 136.5, 137.0,142.7, 166.5, 174.7, 179.8.

Example 404-{5-[4-(3-hydroxypropenyl)-phenyl]-8-oxo-6-thioxo-5,7-diazaspiro[3.4]oct-7-yl}-2-trifluoromethylbenzonitrile,40a [RD120]

To a mixture of 39a (0.05 g, 0.1 mmol) in dichloromethane (2 ml) at −78°C. ms added a solution of diisobutylaluminum hydride in THF (0.11 ml,1M, 0.11 mmol). The mixture was stirred at −78° C. for 3 hours. Afterbeing warmed to room temperature, the mixture was washed with an aqueoussolution of sodium thiosulfate and extracted with ethyl acetate. Theorganic layer was dried over MgSO₄, concentrated and chromatographed(dichloromethane:acetone, 95:5) to yield 40a (0.040 g, 0.089 mmol, 89%)as a white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.57-1.68 (m, 1H), 2.17-2.39 (m, 1H),2.55-2.61 (m, 2H), 2.61-2.67 (m, 2H), 4.39 (d, J=4.7 Hz, 2H), 6.47 (dt,J₁=16.0 Hz, J₂=5.3 Hz, 1H), 6.70 (d, J=16.0 Hz, 1H), 7.29 (d, J=8.3 Hz,2H), 7.59 (d, J=8.3 Hz, 2H), 7.85 (dd, J=8.3 Hz, J₁=1.8 Hz, 1H),7.96-7.98 (m, 2H); ¹³C NMR (CDCl₃, 100 MHz) δ 13.7, 31.5, 63.4, 67.4,110.0, 114.8, 120.5, 121.8 (q, J=272.6 Hz), 127.0 (q, J=4.7 Hz), 127.9,129.2, 130.1, 131.1, 132.1, 133.4 (q, J=33.2 Hz), 135.2, 137.1, 138.4,174.8, 179.9.

Example 41 41-1) 3-[4-(1-cyanocyclobutylamino)-phenyl]-propionic acid,41a (41-1)

Trimethylsilyl cyanide (0.4 g, 4 mmol) was added dropwise to a mixtureof 3-(4-aminophenyl)-propionic acid (0.33 g, 2 mmol), cyclobutanone(0.35 g, 5 mmol) and sodium sulfate (1 g) in 1,4-dioxane (5 ml). Themixture was stirred for 15 hours. After filtration to eliminate sodiumsulfate, the medium was concentrated under vacuum to obtain a brownliquid which was subjected to chromatography (dichloromethane:acetone,50:50) to yield 41a (0.472 g, 1.93 mmol, 97%) as a yellowish solid.

41-2)3-{4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]oct-5-yl]-phenyl}-propionicacid methyl ester, 41b (41-2) [RD128]

A mixture of 1a (0.661 g, 2.9 mmol) and 41a (0.472 g, 1.93 mmol) in dryDMF (2 ml) was stirred at room temperature for 15 hours. To this mixturewere added methanol (10 ml) and HCl aq. (5 ml, 2M). The second mixturewas refluxed for 3 h. After being cooled to room temperature, thereaction mixture was poured into cold water (10 ml) and extracted withethyl acetate (3×30 ml). The organic layer was dried over MgSO₄,concentrated and chromatographed (dichloromethane) to yield 41b (0.582g, 1.19 mmol, 62%) as a white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.60-1.70 (m, 1H), 2.14-2.26 (m, 1H),2.51-2.56 (m, 2H), 2.58-2.67 (m, 2H), 2.71 (t, J=7.8 Hz, 2H), 3.05 (t,J=7.8 Hz, 2H), 3.69 (s, 3H), 7.23 (d, J=8.2 Hz, 2H), 7.41 (d, J=8.2 Hz,2H), 7.85 (dd, J₁=8.3 Hz, J₂=1.8 Hz, 1H), 7.95 (d, J=8.3 Hz, 1H), 7.98(d, J=1.8 Hz, 1H); ¹³C NMR (CDCl₃, 100 MHz) δ 13.7, 30.5, 31.4, 35.1,51.8, 67.5, 109.9, 114.9, 121.9 (q, J=272.7 Hz), 127.1 (q, J=4.7 Hz),129.9, 130.0, 133.2, 132.3, 133.3 (q, J=33.2 Hz), 135.7, 137.2, 142.5,173.1, 174.9, 179.9.

41-3)3-{4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]-phenyl}-propionicacid, 41c (41-3) [RD132]

A mixture of 41b (0.487 g, 1 mmol) in methanol (10 ml) and solution ofsodium hydroxide (10 ml, 2M) was stirred at room temperature for 5hours. Methanol was evaporated. The residue was adjusted to pH=5 by HClaq. (2M) and then extracted with ethyl acetate (3×50 ml). The organiclayer was dried over MgSO₄ and concentrated to dryness to obtain 41c(0.472 g, 0.99 mmol, 99%).

41-4)3-{4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]-phenyl}-propionamide,41d (41-4) [RD133]

To a suspension of 41c (0.094 g, 0.2 mmol) in THF (10 ml) at −5° C. wasadded thionyl chloride (0.019 ml, 0.26 mmol). The medium was stirred at−5° C. for one hour. Then ammonia was bubbled into the mixture. Theexcess of ammonia was condensed by reflux condenser at −78° C. for 30minutes and then was allowed to evaporate. The medium was filtered. Thefiltrate was concentrated and chromatographed (dichloromethane:acetone,70:30) to yield 41d (0.09 g, 0.19 mmol, 95%) as an off-white powder.

¹H NMR (acetone-d₆, 400 MHz) δ 1.52-160 (m, 1H), 2.01-2.09 (m, 1H),2.49-2.58 (m, 4H), 2.61-2.67 (m, 2H), 2.98 (t, J=7.5 Hz, 2H), 6.20 (bs,1H), 6.78 (bs, 1H), 7.31 (d, J=8.2 Hz, 2H), 7.44 (d, J=8.2 Hz, 2H), 8.03(dd, J=8.3 Hz, J₂=1.8 Hz, 1H), 8.15 (d, J=1.8 Hz, 1H), 8.22 (d, J=8.3Hz, 1H); ¹³C NMR (acetone-d₆, 100 MHz) δ 13.4, 30.7, 31.2, 36.4, 67.5,109.0, 114.8, 122.5 (q, J=271.5 Hz), 127.5 (q, J=4.7 Hz), 129.5, 130.0,131.8 (q, J=32.5 Hz), 133.3, 133.8, 135.6, 138.4, 143.2, 171.6, 174.9,178.0.

41-5)3-{4-[7-(4-Cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]-phenyl}-N-methyl-propionamide,41e (41-5) [RD134]

To a suspension of 41c (0.094 g, 0.2 mmol) in THF (10 ml) at −5° C. wasadded thionyl chloride (0.019 ml, 0.26 mmol). The medium was stirred at−5° C. for one hour. Then methylamine was bubbled into the mixture at−5° C. for 30 minutes. The medium was filtered. The filtrate wasconcentrated and chromatographed (dichloromethane:acetone, 75:25) toyield 41e (0.092 g, 0.19 mmol, 95%) as an off-white powder.

¹H NMR (acetone-d₆, 400 MHz) δ 1.51-1.60 (m, 1H), 2.01-2.11 (m, 114),2.48-2.58 (m, 4H), 2.61-2.67 (m, 2H), 2.77 (d, J=4.6 Hz, 3H), 2.98 (t,J=7.5 Hz, 2H), 7.03 (bs, NH), 7.33 (d, J=8.2 Hz, 2H), 7.42 (d, J=8.2 Hz,2H), 8.01 (dd, J₁=8.3 Hz, J₂=1.8 Hz, 1H), 8.13 (d, J=1.8 Hz, 1H), 8.20(d, J=8.3 Hz, 1H); ¹³C NMR (acetone-d₆, 100 MHz) δ 13.4, 25.3, 30.0,31.2, 37.0, 67.6, 109.0, 114.8, 122.5 (q, J=271.5 Hz), 127.4 (q, J=4.7Hz), 129.5, 130.0, 131.9 (q, J=32.5 Hz), 133.3, 133.8, 135.6, 138.4,143.1, 171.7, 175.0, 178.0.

41-6)3-[4-(7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]-phenyl)-N-(2-hydroxyethyl)-propionamide,41f (41-6) [RD135]

To a suspension of 41c (0.094 g, 0.2 mmol) in THF (10 ml) at −5° C. wasadded thionyl chloride (0.019 ml, 0.26 mmol). The medium was stirred at−5° C. for one hour. Then 2-aminoethanol (0.0183 g, 0.03 mmol) was addedinto the mixture at −5° C. After stirring of an additional 30 minutes,the medium was filtered. The filtrate was concentrated andchromatographed (dichloromethane:acetone, 50:50) to yield 41f (0.093 g,0.18 mmol, 90%) as an off-white powder.

¹H NMR (acetone-d₆, 400 MHz) δ 1.51-161 (m, 1H), 2.01-2.11 (m., 1H),2.49-2.66 (m, 6H), 2.99 (t, J=7.5 Hz, 2H), 3.27 (dd, J_(J)==5.6 Hz, 3H),3.51 (dd, J₁=11.2 Hz, J₂=5.6 Hz, 2H), 3.87 (bs, OH), 7.20 (bs, NH), 7.33(d, J=8.2 Hz, 2H), 7.43 (d, J=8.2 Hz., 2H), 8.02 (dd, J=8.3 Hz, J₂=1.8Hz, 1H), 8.14 (d, J=1.8 Hz, 1H), 8.22 (d, J=8.3 Hz, 1H); ¹³C NMR(acetone-d₆, 100 MHz) δ 13.4, 31.0, 31.2, 37.1, 42.0, 61.2, 67.6, 109.0,114.8, 122.5 (q, J=271.5 Hz), 127.4 (q, J=4.7 Hz), 129.6, 130.0, 131.9(q, J=32.5 Hz), 133.3, 133.8, 135.6, 138.4, 143.0, 171.9, 175.0, 178.1.

42-1) 4-[4-(1-Cyanecyclobutylamino)-phenyl]-butyric acid, 42a

Trimethylsilyl cyanide (0.50 g, 5 mmol) was added dropwise to a mixtureof 4-(4-aminophenyl)-butyric acid (0.537 g, 3 mmol), cyclobutanone (0.35g, 5 mmol) and sodium sulfate (1 g) in 1,4-dioxane (10 ml). The mixturewas stirred for 15 hours. After filtration to eliminate sodium sulfate,the medium was concentrated under vacuum to obtain a brown liquid whichwas subjected to chromatography (dichloromethane:acetone, 50:50) toyield 42a (0.665 g, 2.58 mmol, 86%) as a yellowish solid.

42-2) 4-{4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-dazaspiro[3.4]oct-5-yl]phenyl}-butyric acid methyl ester, 42b [RD129]

A mixture of 1a (0.547 g, 2.4 mmol) and 42a (0.342 g, 1.5 mmol) in dryDMF (2 ml) was stirred at room temperature for 15 hours. To this mixturewere added methanol (10 ml) and HCl aq. (5 ml, 2M). The second mixturewas refluxed for 3 h. After being cooled to room temperature, thereaction mixture was poured into cold water (10 ml) and extracted withethyl acetate (3×30 ml). The organic layer was dried over MgSO₄,concentrated and chromatographed (dichloromethane) to yield 42b (0.594g, 1.18 mmol, 79%) as a white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.60-1.70 (m, 1H), 1.98-2.07 (m, 2H),2.14-2.26 (m, 1H), 2.40 (t, J=7.4 Hz, 2H), 2.52-2.60 (m, 2H), 2.62-2.68(m, 2H), 2.74 (t, J=7.4 Hz, 2H), 3.68 (s, 3H), 7.22 (d, J=8.2 Hz, 2H),7.38 (d, J=8.2 Hz, 2H), 7.86 (dd, J=8.3 Hz, J₂=1.8 Hz, 1H), 7.95 (d,J=8.3 Hz, 1H), 7.98 (d, J=1.8 Hz, 1H); NMR (CDCl₃, 100 MHz) δ 13.7,26.1, 31.4, 33.5, 34.8, 51.7, 67.5, 109.9, 114.9, 121.9 (q, J=272.7 Hz),127.1 (q, J=4.7 Hz), 129.7, 130.1, 132.3, 133.0, 133.3 (q, J=33.2 Hz),135.2, 137.2, 143.5, 173.8, 175.0, 179.9.

42-3)4-{4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]-phenyl}-butyricacid, 42c [RD141]

A mixture of 42b (0.501 g, 1 mmol) in methanol (10 ml) and solution ofsodium hydroxide (10 ml, 2M) was stirred at room temperature for 5hours. The methanol was evaporated. The residue was adjusted to pH=5 byHCl aq. (2M) and then, the medium was extracted with ethyl acetate (3×50ml). The organic layer was dried over MgSO₄ and concentrated to drynessto obtain 42c (0.482 g, 0.99 mmol, 99%), the structure of which isillustrated in Formula 5.

¹H NMR (CDCl₃, 400 MHz) δ 1.60-1.70 (m, 1H), 1.98-2.07 (m, 2H),2.14-2.26 (m, 1H), 2.45 (t, J=7.3 Hz, 2H), 2.51-2.59 (m, 2H), 2.62-2.68(m, 2H), 2.77 (t, J=7.3 Hz, 2H), 7.23 (d, J=8.1 Hz, 2H), 7.40 (d, J=8.1Hz, 2H), 7.85 (dd, J=8.3, 1.8 Hz, 1H), 7.95 (d, J=8.3 Hz, 1H), 7.97 (d,J=1.8 Hz, 1H); ¹³C NMR (CDCl₃, 100 MHz) δ 13.7, 25.9, 31.4, 33.4, 34.7,67.5, 109.9, 114.9, 121.9 (q, J=272.6 Hz), 127.1 (q, J=4.7 Hz), 129.8,130.1, 132.3, 133.0, 133.4 (q, J=33.1 Hz), 135.2, 137.2, 143.3, 174.9,178.9, 179.9.

42-4)4-{4-[7-(4-Cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]-phenyl}-butyramide,42d [RD130]

To a suspension of 42c (0.097 g, 0.2 mmol) in THF (10 ml) at −5° C. wasadded thionyl chloride (0.019 ml, 0.26 mmol). The medium was stirred at−5° C. for one hour. Then ammonia was bubbled into the mixture. Theexcess of ammonia was condensed by reflux condenser at −78° C. for 30minutes and then was allowed to evaporate. The medium was filtered. Thefiltrate was concentrated and chromatographed (dichloromethane:acetone,70:30) to yield 42d (0.093 g, 0.19 mmol, 95%) as an off-white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.57-1.70 (m, 1H), 2.00-2.08 (m, 2H),2.16-2.25 (m, 1H), 2.31 (t, J=7.3 Hz, 2H), 2.51-2.59 (m, 2H), 2.62-2.68(m, 2H), 2.77 (t, J=7.3 Hz, 2H), 5.56 (bs, 1H), 5.65 (bs, 1H), 7.22 (d,J=8.2 Hz, 2H), 7.39 (d, J=8.2 Hz, 2H), 7.85 (dd, J₁=8.3 Hz, J₂=1.8 Hz,1H), 7.95 (d, J=8.3 Hz, 1H), 7.97 (d, J=1.8 Hz, 1H); ¹³C NMR (CDCl₃, 100MHz) δ 13.7, 26.5, 31.4, 34.8, 35.0, 67.5, 109.9, 114.9, 121.9 (q,J=272.7 Hz), 127.1 (q, J=4.7 Hz), 129.8, 130.1, 132.2, 133.0, 133.3 (q,J=33.2 Hz), 135.2, 137.2, 143.5, 173.8, 174.9, 179.9.

42-5)4-{4-[7-(4-Cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]phenyl}-N-methyl-butyramide,42e [RD131]

To a suspension of 42c (0.097 g, 0.2 mmol) in THF (10 ml) at −5° C. wasadded thionyl chloride (0.019 ml, 0.26 mmol). The medium was stirred at−3° C. for one hour. Then methylamine was bubbled into the mixture at−5° C. for 30 minutes. The medium was filtered. The filtrate wasconcentrated and chromatographed (dichloromethane:acetone, 75:25) toyield 42e (0.095 g, 0.19 mmol, 95%) as an off-white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.52-1.64 (m, 1H), 1.94-2.01 (m, 2H),2.10-2.17 (m, 1H), 2.20 (t, J=7.3 Hz, 2H), 2.46-2.62 (m., 4H), 2.69 (t,J=7.3 Hz, 2H), 2.73 (d, J=4.7 Hz, 3H), 6.09 (bs, 1H), 7.16 (d, J=8.2 Hz,2H), 7.33 (d, J=8.2 Hz, 2H), 7.82 (dd, J₁=8.3 Hz, J₂=1.8 Hz, 1H), 7.91(d, J=8.3 Hz, 1H), 7.94 (d, J=1.8 Hz, 1H); ¹³C NMR (CDCl₃, 100 MHz) δ13.7, 26.2, 26.8, 31.4, 35.0, 35.7, 67.5, 109.7, 114.9, 121.9 (q,J=272.7 Hz), 127.1 (q, J=4.7 Hz), 129.7, 130.0, 132.3, 133.8, 133.3 (q,J=33.2 Hz), 135.2, 137.3, 143.7, 173.3, 174.9, 179.8.

42-6)N-(4-{4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]phenyl}-butanoyl)-methanesulfonamide,42f [RD157]

A mixture of4-{4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]phenyl}butanoicacid (42c) (0.049 g, 0.1 mmol), 2,4,6-trichlorobenzoyl chloride (0.244g, 1 mmol), 4-dimethylaminopyridine (0.122 g, 1 mmol) andmethanesulfonamide (0.019 g, 0.2 mmol) in dichloromethane was stirred atroom temperature for 20 hours. The mixture was concentrated andchromatographed (dichloromethane:acetone, 80:20) to yieldN-(4-{4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]phenyl}-butanoyl)-methanesulfonamide(42f) [RD 157] (0.053 g, 0.094 mmol, 94%), the structure of which isillustrated in Formula 8, as a white powder.

¹H NMR (acetone-d₆, 400 MHz) δ 1.51-160 (m, 1H), 1.96-2.11 (m, 3H), 2.49(t, J=7.3 Hz, 2H), 2.51-2.57 (m, 2H), 2.61-2.67 (in., 2H), 2.75 (t,J=7.5 Hz, 2H), 2.94 (bs, 1H), 3.24 (s, 3H), 7.33 (d, J=8.3 Hz, 2H), 7.43(d, J=8.2 Hz, 2H), 8.02 (dd, J=8.3, 1.6 Hz, 1H), 8.02 (d, J=1.6 Hz, 1H),8.21 (d, J=8.3 Hz, 1H); ¹³C NMR (acetone-d₆, 100 MHz) δ 13.4, 25.8,31.2, 34.3, 35.2, 40.6, 67.6, 109.0, 114.8, 122.5 (q, J=271.5 Hz), 127.5(q, J=4.9 Hz), 129.6, 130.1, 131.9 (q, J=33.6 Hz), 133.3, 133.9, 135.6,138.4, 143.1, 171.9, 175.0, 180.5.

42-7)N-methyl-4-{4-[7-(4-cyano-3-trifluoromethylphenyl)-6,8-dioxo-5,7-diazaspiro[3.4]oct-5-yl]-phenyl}butyramide,42g [RD158]

Hydrogen peroxide (30%, 0.4) was added dropwise to a solution ofN-methyl-4-{4-[7-(4-Cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]phenyl}butanamide(42e) (0.032 g, 0.064 mmol) in glacial acetic acid (0.5 ml). The mixturewas stirred at room temperature for 5 hours and then washed with waterand extracted with ethyl acetate. The organic layer was dried overmagnesium sulfate, concentrated and chromatographed(dichloromethane:acetone, 80:20) to yieldN-methyl-4-{4-[7-(4-cyano-3-trifluoromethylphenyl)-6,8-dioxo-5,7-diazaspiro[3.4]oct-5-yl]-phenyl}butyramide(42g) [RD158] (0.029 g, 0.06 mmol, 94%), the structure of which isillustrated in Formula 9, as a white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.63-1.71 (m, 1H), 1.93-2.04 (m, 2H),2.18-2.27 (m, 3H), 2.44-2.53 (m, 2H), 2.57-2.65 (m, 2H), 2.70 (t, J=7.3Hz, 2H), 2.79 (d, J=4.8 Hz, 3H), 5.79 (bs, 1H), 7.21 (d, J=8.2 Hz, 2H),7.34 (d, J=8.2 Hz, 2H), 7.92 (d, J=8.4 Hz, 1H), 8.03 (dd, J=8.3, 1.8 Hz,1H), 8.18 (d, J=1.8 Hz, 1H).

Example 43 43-1) 4-(4-aminophenyl)-piperazine-1-carboxylic acidtert-butyl ester, 43a

A mixture of 4-iodoaniline (0.654 g, 3 mmol), piperazine-1-carboxylicacid tert-butyl ester (0.67 g, 3.6 mmol), potassium phosphate (1.272 g,6 mmol), ethylene glycol (0.33 ml) and copper iodide (0.03 g, 0.15 mmol)in 2-propanol (3 ml) was placed under argon in a sealed-tube and heatedto 80° C. for 30 hours. After being cooled to room temperature, themedium was washed with water (50 ml) and extracted with ethyl acetate(100 ml). The organic layer was dried over MgSO₄, concentrated andchromatographed (dichloromethane:acetone, 70:30) to yield 43a (0.36 g,1.3 mmol, 43%) as a yellow powder.

43-2) 4-[4-(1-cyanocyclobutylamino)phenyl]-piperazine-1-carboxylic acidtert-butyl ester, 43b

Trimethylsilyl cyanide (0.3 g, 3 mmol) was added dropwise to a mixtureof 43a (0.415 g, 1.5 mmol), cyclobutanone (0.21 g, 3 mmol) and sodiumsulfate (1 g) in dichloromethane (5 ml). The mixture was stirred for 15hours. After filtration to eliminate sodium sulfate, the medium wasconcentrated under vacuum to obtain a brown liquid which was subjectedto chromatography (dichloromethane:acetone, 75:25) to yield 43b (0.448g, 1.26 mmol, 84%) as a yellow solid.

43-3)4-{4-[7-(4-cyano-3-trifluoromethylphenyl)-8-imino-6-thioxo-5,7-diazaspiro[3.4]oct-5-yl]-phenyl}-piperazine-1-carboxylicacid tert-butyl ester, 43c [RD139] and4-{4-[7-(4-cyano-3-trifluoromethylphenyl)-8-(4-cyano-3-trilluoromethyl-phenylthiocarbamoylimino)-6-thioxo-5,7-diazaspiro[3.4]oct-5-yl]-phenyl}-piperazine-1-carboxylic acid tert-butyl ester, 43d [RD140]

A mixture of 1a (0.228 g, 1 mmol) and 43b (0.472 g, 0.63 mmol) in dryDMF (1 ml) was stirred at room temperature for 20 hours. The mixture wasconcentrated and chromatographed (dichloromethane:acetone, 90:10) toyield 43c (0.173 g, 0.296 mmol, 47%), the structure of which isillustrated in Formula 10, as a off-white powder and 43d (0.169 g, 0.21mmol, 33%), the structure of which is illustrated in Formula 11, as ayellow powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.48, (s, 9H), 1.57-1.67 (m, 1H), 2.01-2.09(m, 1H), 2.59-2.70 (m, 4H), 3.25 (t, J=5.1 Hz, 4H), 3.59 (t, J=4.9 Hz,4H), 7.02 (d, J=8.9 Hz, 2H), 7.20 (d, J=8.9 Hz, 2H), 7.81 (d, J=7.4 Hz,1H), 7.93 (s, 1H), 7.97 (d, J=8.1 Hz, 1H).

¹H NMR (CDCl₃, 400 MHz) δ 1.48, (s, 9H), 1.57-1.64 (m, 1H), 2.01-2.10(m, 1H), 2.60-2.89 (m, 4H), 3.24 (t, J=5.1 Hz, 4H), 3.57 (t, J=4.9 Hz,4H), 7.02 (d, J=8.9 Hz, 2H), 7.20 (d, J=8.9 Hz, 2H), 7.54-7.98 (m, 4H),7.97 (d, J=8.1 Hz, 1H).

43-4)4-[8-Oxo-5-(4-piperazin-1-yl-phenyl)-6-thioxo-5,7-diazaspiro[3,4]oct-7-yl]-2-trifluoromethylbenzonitrile,43e [RD137]

A mixture of 43c (0.117 g, 0.2 mmol), methanol (5 ml) and HCl aq. (2 ml,2M) was refluxed for 2 hours. After being cooled to room temperature,the reaction mixture was poured into cold water (10 ml) and extractedwith ethyl acetate (3×30 ml). The organic layer was dried over MgSO₄,concentrated and chromatographed (dichloromethane:acetone, 50:50 andthen methanol:acetone, 50:50) to yield 43e (0.089 g, 0.184 mmol, 92%) asa white powder.

¹H NMR (CD₃OD, 400 MHz) δ 1.51-1.61 (m, 1H), 2.01-2.11 (m, 1H),2.48-2.59 (m, 4H), 2.90-2.97 (m, 4H), 3.25-3.30 (m, 4H), 7.03 (d, J=8.9Hz, 2H), 7.16 (d, J=8.9 Hz, 2H), 7.86 (dd, =8.3 Hz, J=1.8 Hz, 1H), 8.02(d, J=8.3 Hz, 1H), 8.07 (d, J=1.8 Hz, 1H); ¹³C NMR (CD₃OD, 100 MHz) δ13.2, 30.9, 45.1, 48.9, 67.5, 108.9, 114.8, 115.9, 122.3 (q, J=271.7Hz), 126.4, 127.3 (q, J=4.7 Hz), 130.4, 132.2 (q, J=33.2 Hz), 133.0,135.4, 138.1, 152.1, 175.4, 180.4.

43-5)4-{5-[4-(4-methanesulfonylpiperazin-1-yl)-phenyl]-8-oxo-6-thioxo-5,7-diazaspiro[3.4]oct-7-yl}-2-trifluoromethylbenzonitrile,43f [RD138]

A mixture of 43e (0.049 g, 0.1 mmol), methanesulfonyl chloride (0.012ml, 0.15 mmol) and triethylamine (0.15 ml) in dichloromethane wasstirred at room temperature for 5 hours. The medium was filtered. Thefiltrate was concentrated and chromatographed (dichloromethane: acetone,95:3) to yield 43f (0.042 g, 0.074 mmol, 74%) as a white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.62-1.70 (m, 1H), 2.14-2.23 (m, 1H),2.51-2.58 (m, 2H), 2.61-2.67 (m, 214), 2.84 (s, 3H), 3.39 (s, 8H), 7.05(d, J=8.9 Hz, 2H), 7.20 (d, J=8.9 Hz, 2H), 7.84 (dd, J=8.3 Hz, J₂=1.8Hz, 1H), 7.95 (d, J=8.3 Hz, 1H), 7.97 (d, J=1.8 Hz, 1H); ¹³C NMR (CDCl₃,100 MHz) δ 13.7, 31.4, 34.6, 45.7, 48.4, 67.5, 109.8, 114.9, 117.0,121.9 (q, J=272.7 Hz), 126.8, 127.1 (q, J=4.7 Hz), 130.7, 132.3, 133.4(q, J=33.2 Hz), 135.2, 137.3, 151.1, 175.0, 180.2.

Example 44 44-1)3-{4-[7-(4-Cyano-3-trifluoromethyl-phenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]-phenyl}-acrylicacid, 44a

A mixture of 39a (0.025 g, 0.05 mmol) in methanol (2 ml) and solution ofsodium hydroxide (2 ml, 2M) was stirred at room temperature for 5 hours.Methanol was evaporated. The residue was adjusted to pH=5 by HCl aq.(2M) and then extracted with ethyl acetate (3×50 ml). The organic layerwas dried over MgSO₄ and concentrated to dryness to obtain 44a (0.02 g,0.042 mmol, 85%).

44-2)3-{4-[7-(4-Cyano-3-trifluoromethyl-phenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]-phenyl}-acrylamide,44b [RD119]

To a suspension of 44b (0.02 g, 0.042 mmol) in THF (1 ml) at −5° C. wasadded thionyl chloride (0.007 ml, 0.1 mmol). The medium was stirred at−5° C. for one hour. Then ammonia was bubbled into the mixture. Theexcess of ammonia was condensed by reflux condenser at −78° C. for 30minutes and then was allowed to evaporate. The medium was filtered. Thefiltrate was concentrated and chromatographed (dichloromethane:acetone,70:30) to yield 44b (0.014 g, 0.03 mmol, 71%) as an off-white powder.

¹H NMR (DMSO-d₆, 400 MHz) δ 1.49-1.52 (m, 1H), 1.88-1.93 (m, 1H),2.37-2.46 (m, 2H), 2.57-2.62 (m, 2H), 6.66 (d, J=15.9 Hz, 1H), 7.16 (bs,1H), 7.43 (d, J=8.3 Hz, 2H), 7.47 (d, J=15.9 Hz, 1H), 7.58 (bs, 1H),8.03 (dd, J=8.3 Hz, J_(Z)=1.8 Hz, 1H), 8.23 (d, J=1.8 Hz, 1H), 8.34 (d,J=8.3 Hz, 1H).

Example 45 RD145

Trimethylsilyl cyanide (0.4 g, 4 mmol) was added dropwise to a mixtureof 4-methanesulfonylphenylamine hydrochloride (0.415 g, 2 mmol),cyclobutanone (0.28 g, 4 mmol) and sodium sulfate (1 g) in DMF (3 ml).The mixture was stirred for 15 hours at 120° C. After filtration toremove the sodium sulfate, the filtrate was washed with brine andextracted with ethyl acetate. The organic layer was concentrated andchromatographed (dichloromethane:acetone, 90:10) to yield1-(4-methanesulfonylphenylamino)cyclobutanecarbonitrile (45a) (0.116 g,0.44 mmol, 22%) as a yellowish solid. 4-methanesulfonylphenylamine(0.201 g, 1.17 mmol, 59%) was also recovered.

A mixture of 4-isothiocyanato-2-trifluoromethylbenzonitrile (1a)(0.0.141 g, 0.62 mmol) and1-(4-methanesulfonylphenylamino)cyclobutanecarbonitrile (45a) (0.11 g,0.42 mmol) in dry DMF (2 ml) was stirred at room temperature for 3 days.To this mixture were added methanol (10 ml) and aq. 2N HCl (5 ml). Thesecond mixture was refluxed for 3 h. After being cooled to roomtemperature, the reaction mixture was poured into cold water (10 ml) andextracted with ethyl acetate (3×30 ml). The organic layer was dried overMgSO₄, concentrated and chromatographed (dichloromethane:acetone, 97:3)to yield4-[5-(4-methanesulfonylphenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]oct-7-yl]-2-trifluoromethylbenzonitrile(45b) [RD145] (0.031 g, 0.065 mmol, 15%), the structure of which isillustrated in Formula 14, as a white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.63-1.72 (m, 1H), 2.21-2.28 (m, 1H),2.46-2.54 (m, 2H), 2.68 2.74 (m, 2H), 3.16 (s, 3H), 7.57 (d, J=8.3 Hz,2H), 7.85 (dd, J=8.3, 1:8 Hz, 1H), 7.97 (d, J=1.8 Hz, 1H), 7.98 (d,J=8.3 Hz, 1H), 8.17 (d, J=8.3 Hz, 2H); ¹³C NMR (CDCl₃, 100 MHz) δ 13.6,31.8, 44.4, 67.5, 110.2, 114.8, 122.4 (q, J=271.5 Hz), 127.0 (q, J=4.9Hz), 129.4, 131.4, 132.1, 133.6 (q, J=33.3 Hz), 135.3, 136.8, 140.3,141.8, 174.4, 179.9.

Example 46

Trimethylsilyl cyanide (0.69 g, 7 mmol) was added dropwise to a mixtureof 4-aminophenylacetic acid (0.755 g, 5 mmol) and cyclobutanone (0.49 g,7 mmol) in dioxane (20 ml). The mixture was stirred for 8 hours at 80°C. The mixture was concentrated and chromatographed(dichloromethane:acetone, 60:40) to yield[4-(1-cyanocyclobutylamino)phenyl]acetic acid (46a) (1.138 g, 4.95 mmol,99%) as a white solid.

46-1) RD146

A mixture of 4-isothiocyanato-2-trifluoromethylbenzonitrile (1a) (0.638g, 2.8 mmol) and [4-(1-cyanocyclobutylamino)phenyl]acetic acid (46a)(0.46 g, 2.0 mmol) in DMF (5 ml) was stirred at room temperature for 15hours. To this mixture were added methanol (20 ml) and aq. 2N HCl (10ml). The second mixture was refluxed for 1 h. After being cooled to roomtemperature, the reaction mixture was poured into cold water (10 ml) andextracted with ethyl acetate (3×50 ml). The organic layer was dried overMgSO₄, concentrated and chromatographed (dichloromethane pure and thendichloromethane:acetone, 95:5) to yield{4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]phenyl}aceticacid methyl ester (46b) [RD146] (0.532 g, 1.124 mmol, 56%), thestructure of which is illustrated in Formula 15, as a white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.60-1.69 (m, 1H), 2.15-2.25 (m, 1H),2.50-2.58 (m, 2H), 2.61-2.66 (m, 2H), 3.72 (bs, 5H), 7.27 (d, J=8.3 Hz,2H), 7.50 (d, J=8.3 Hz, 2H), 7.84 (dd, J=8.3, 1.8 Hz. 1H). 7.94 (d,J=8.2 Hz, 1H), 7.97 (d, J=1.6 Hz, 1H); ¹³C NMR (CDCl₃, 100 MHz) δ 13.7,31.4, 44.7, 52.3, 67.4, 109.9, 114.9, 122.0 (q, J=272.5 Hz), 127.0 (q,J=4.9 Hz), 130.0, 131.1, 132.3, 133.0 (q, J=33.3 Hz), 134.1, 135.2,135.9, 137.2, 171.4, 174.9, 179.9.

46-2) RD147

A mixture of{4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]phenyl}aceticacid methyl ester (46b) (0.095 g, 0.2 mmol) and a solution of sodiumhydroxide (1 ml, 2M) in methanol (2 ml) was stirred at room temperaturefor 2 hours. The methanol was evaporated. The residue was adjusted to pH5 by aq. 2M HCl and then the mixture was extracted with ethyl acetate(3×10 ml). The organic layer was dried over MgSO₄ and concentrated todryness to obtain{4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]phenyl}aceticacid (46c) [RD147] (0.087 g, 0.19 mmol, 95%), the structure of which isillustrated in Formula 16.

¹H NMR (CDCl₃, 400 MHz) δ 1.60-1.69 (m, 1H), 2.15-2.25 (m, 1H),2.50-2.64 (m, 4H), 3.73 (s, 2H), 7.26 (d, J=8.3 Hz, 2H), 7.51 (d, J=8.3Hz, 2H), 7.84 (dd, J=8.3, 1.8 Hz, 1H), 7.95 (d, J=8.2 Hz, 1H), 7.96 (d,J=1.6 Hz, 1H); ¹³C NMR (CDCl₃, 100 MHz) δ 13.7, 31.4, 40.2, 40.8, 67.4,109.9, 114.9, 122.0 (q, J=272.5 Hz), 127.0 (q, J=4.9 Hz), 129.9, 131.2,132.3, 133.3 (q, J=33.3 Hz), 133.9, 135.2, 136.1, 137.2, 174.1, 174.9,179.9.

46-3) RD148

Thionyl chloride (0.238 g, 2 mmol) was added dropwise to a mixture of{4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]phenyl}aceticacid (46c) (0.357 g, 0.777 mmol) in THF (5 ml) cooled to 0° C. Themixture was stirred for 1 hour at room temperature and then ammonia wasbubbled into the mixture. The excess ammonia was condensed by a refluxcondenser at −78° C. for 30 minutes and then was allowed to evaporate.The medium was filtered and the filtrate was concentrated andchromatographed (dichloromethane:acetone, 70:30) to yield2-{4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]phenyl}acetamide(46d) [RD148] (0.345 g, 0.75 mmol, 97%), the structure of which isillustrated in Formula 17, as an off-white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.62-1.66 (m, 1H), 2.18.2.23 (m, 1H),2.49-2.55 (m, 2H), 2.61-2.66 (m, 2H), 3.63 (s, 2H), 5.91 (bs, 1H), 6.10(bs, 1H), 7.27 (d, J=8.1 Hz, 2H), 7.50 (d, J=8.1 Hz, 2H), 7.83 (dd,J=8.3, 1.8 Hz, 1H), 7.95 (d, J=8.2 Hz, 1H), 7.96 (d, J=1.6 Hz, 1H); ¹³CNMR (CDCl₃, 100 MHz) δ 13.7, 31.5, 42.5, 67.4, 109.9, 114.9, 121.9 (q,J=272.4 Hz), 127.1 (q, J=4.9 Hz), 130.2, 131.1, 132.2, 133.3 (q, J=33.3Hz), 134.1, 135.2, 136.8, 137.2, 172.8, 174.8, 180.0.

46-4) RD149

Thionyl chloride (0.238 g, 2 mmol) was added dropwise to a mixture of{4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]phenyl}aceticacid (46c) (0.357 g, 0.777 mmol) in THF (5 ml) cooled to 0° C. Themixture was stirred for 1 hour at room temperature and then methylamine(0.5 ml) was added into the mixture. The mixture was stirred for anadditional 2 hours. The medium was filtered and the filtrate wasconcentrated and chromatographed (dichloromethane:acetone, 80:20) toyieldN-methyl-2-{4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]phenyl}acetamide(46e) [RD149] (0.348 g, 0.738 mmol, 95%), the structure of which isillustrated in Formula 18, as an off-white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.61-1.70 (m, 1H), 2.17-2.31 (m, 1H),2.50-2.56 (m, 2H), 2.61-2.68 (m, 2H), 2.82 (d, J=4.8 Hz, 3H), 3.62 (s,2H), 7.27 (d, J=8.3 Hz, 2H), 7.50 (d, J=8.3 Hz, 2H), 7.84 (dd, J=8.3,1.8 Hz, 1H), 7.95 (d, J=8.2 Hz, 1H), 7.96 (d, J=1.6 Hz, 1H); ¹³C NMR(CDCl₃, 100 MHz) δ 13.7, 26.6, 31.5, 43.1, 67.4, 110.0, 114.9, 122.0 (q,J=272.5 Hz), 127.1 (q, J=4.9 Hz), 130.2, 131.0, 132.2, 133.3 (q, J=33.3Hz), 134.1, 135.2, 137.0, 137.1, 170.1, 174.8, 179.9.

Example 47N-{4-[3-(4-cyano-3-trifluoromethylphenyl)-5,5-dimethyl-4-oxo-2-thioxo-imidazolidin-1-yl]phenyl}methanesulfonamide(47a) [RD150]

A mixture of4-[3-(4-aminophenyl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile(2d) (0.02 g, 0.05 mmol), methanesulfonyl chloride (0.009 g, 0.075 mmol)and pyridine (0.006 g, 0.075 mmol) in dichloromethane (1 ml) was stirredat room temperature for 15 hours. The medium was washed with water (2ml) and extracted with ethyl acetate (5 ml). The organic layer was driedover MgSO₄, concentrated and chromatographed (HPLC, alumina column) toyieldN-{4-[3-(4-cyano-3-trifluoromethyl-phenyl)-5,5-dimethyl-4-oxo-2-thioxo-imidazolidin-1-yl]phenyl}methanesulfonamide(47a) [RD150] (0.009 g, 0.018 mmol, 36%), the structure of which isillustrated in Formula 2, as a white powder.

¹H NMR (DMSO-d₆, 400 MHz) δ 1.46 (s, 6H), 3.07 (s, 3H), 7.32 (s, 4H),8.05 (dd, J=8.2, 1.2 Hz, 1H), 8.26 (d, J=1.2 Hz, 1H), 8.35 (d, J=8.2 Hz,1H), 10.08 (bs, 1H); ¹³C NMR (DMSO-d₆, 100 MHz) δ 23.3, 40.4, 66.7,109.0, 115.5, 119.9, 122.6 (q, J=272.2 Hz), 128.5 (q, J=4.7 Hz), 130.8,131.2, 131.5 (q, J=32.3 Hz), 134.5, 136.6, 138.6, 139.5, 175.4, 180.4.

Example 48N-{4-[3-(4-cyano-3-trifluoromethylphenyl)-5,5-dimethyl-4-oxo-2-thioxo-imidazolidin-1-yl]phenyl}acetamide,48a, [RD151]

A mixture of4-[3-(4-aminophenyl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile(2d) [RD9] (0.008 g, 0.02 mmol), acetyl chloride (0.004 g, 0.03 mmol)and triethylamine (0.003 g, 0.03 mmol) in dichloromethane (1 ml) wasstirred at 0° C. for 2 hours. The mixture was concentrated andchromatographed (dichloromethane:acetone, 90:10) to yieldN-{4-[3-(4-cyano-3-trifluoromethylphenyl)-5,5-dimethyl-4-oxo-2-thioxo-imidazolidin-1-yl]phenyl}acetamide,48a, [RD151] (0.007 g, 0.016 mmol, 80%), the structure of which isillustrated in Formula 3, as a white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.58 (s, 6H), 2.21 (s, 3H), 7:24 (d, J=8.6 Hz,2H), 7.48 (bs, 1H), 7.69 (d, J=8.6 Hz, 2H), 7.83 (dd, J=8.2, 1.9 Hz,1H), 7.96 (d, J=1.2 Hz, 1H), 7.97 (d, J=8.2 Hz, 1H); ¹³C NMR (CDCl₃, 100MHz) δ 23.6, 53.4, 66.4, 110.0, 114.8, 120.7, 122.6 (q, J=272.2 Hz),127.1 (q, J=4.7 Hz), 129.1, 130.2, 132.2, 133.5 (q, J=32.3 Hz), 135.2,137.1, 139.2, 168.1, 175.0, 180.0.

Example 49

Concentrated sulfuric acid was slowly added to a mixture of4-aminobenzoic acid (4 g, 29.2 mmol) in methanol cooled to 0° C. Afterthe addition, the mixture was stirred at room temperature for 5 hours.The mixture was washed with a saturated solution of sodium bicarbonateand extracted with ethyl acetate. The organic layer was dried over MgSO₄and concentrated under vacuum to obtain 4-aminobenzoic acid methyl ester(49a) (4.22 g, 27.9 mmol, 96%) as an off-white solid.

A mixture of 4-aminobenzoic acid methyl ester (0.32 g, 2.12 mmol),acetonecyanohydrin (3 ml) and sodium sulfate (1 g) was refluxed for 15hours. After filtration to remove the sodium sulfate, the filtrate waswashed with brine and extracted with ethyl acetate. The organic layerwas concentrated and chromatographed (dichloromethane:acetone, 60:40) toyield 4-[(cyanodimethylmethyl)-amino]-benzoic acid methyl ester (49b)(0.398 g, 1.95 mmol, 92%) as a white solid.

49-1) RD152

A mixture of 4-isothiocyanato-2-trifluoromethylbenzonitrile (1a) (0.228g, 1 mmol) and 4-[(cyanodimethylmethyl)-amino]-benzoic acid methyl ester(49b) (0.14 g, 0.64 mmol) in DMF (2 ml) was heated under microwaveirradiation at 60° C. for 12 hours. To this mixture were added methanol(6 ml) and aq. 2N HCl (2 ml). The second mixture was refluxed for 4 h.After being cooled to room temperature, the reaction mixture was pouredinto cold water (10 ml) and extracted with ethyl acetate (3×30 ml). Theorganic layer was dried over MgSO₄, concentrated and chromatographed(dichloromethane; dichloromethane:acetone, 75:25) to yield4-[3-(4-cyano-3-trifluoromethylphenyl)-5,5-dimethyl-4-oxo-2-thioxo-imidazolidin-1-yl]benzoicacid methyl ester (49c) [RD152] (0.18 g, 0.4 mmol, 63%), the structureof which is illustrated in Formula 19, as a white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.60 (s, 6H), 3.95 (s, 3H), 7.40 (d, J=8.6 Hz,2H), 7.84 (dd, J=8.2, 1.9 Hz, 1H), 7.96 (d, J=1.2 Hz, 1H), 7.97 (d,J=8.2 Hz, 1H), 8.21 (d, J=8.6 Hz, 2H); ¹³C NMR (CDCl₃, 100 MHz) δ 23.8,52.6, 66.6, 110.3, 114.8, 121.9 (q, J=272.7 Hz), 127.1 (q, J=4.7 Hz),129.8, 131.2, 131.4, 132.2, 133.5 (q, J=32.3 Hz), 135.3, 137.0, 139.2,165.9, 174.7, 179.7.

49-2) RD153

A mixture of4-[3-(4-cyano-3-trifluoromethylphenyl)-5,5-dimethyl-4-oxo-2-thioxo-imidazolidin-1-yl]benzoicacid methyl ester (49c) (0.02 g, 0.0435 mmol) and methylamine (2 mldistilled from its 40% aqueous solution) was kept at −20° C. for 15hours. After evaporation of the methylamine, the mixture waschromatographed (dichloromethane:acetone, 80:20) to yield4-[3-(4-cyano-3-trifluoromethylphenyl)-5,5-dimethyl-4-oxo-2-thioxo-imidazolidin-1-yl]-N-methylbenzamide(49d) [RD153] (0.01 g, 0.0224, 51%), the structure of which isillustrated in Formula 20. The ester4-[3-(4-cyano-3-trifluoromethylphenyl)-5,5-dimethyl-4-oxo-2-thioxo-imidazolidin-1-yl]benzoicacid methyl ester (49c) (0.08 g, 0.0179 mmol, 41%) was also recovered.

¹H NMR (Acetone-d₆, 400 MHz) δ 1.60 (s, 6H), 2.90 (d, J=4.6 Hz, 3H),7.48 (d, J=8.6 Hz, 2H), 7.80 (bs, 1H), 7.99 (d, J=8.6 Hz, 2H), 8.06 (dd,J=8.2, 1.8 Hz, 1H), 8.18 (d, J=1.8 Hz, 1H), 8.25 (d, J=8.2 Hz, 1H); ¹³CNMR (Acetone-d₆, 100 MHz) δ 23.8, 54.0, 66.5, 110.3, 114.8, 121.9 (q,J=272.7 Hz), 127.1 (q, J=4.7 Hz), 128.2, 129.9, 133.5 (q, J=32.3 Hz),135.7, 135.8, 138.2, 138.3, 139.2, 166.0, 174.9, 179.7.

Example 50 50-1) RD154

A mixture of4-[8-(4-hydroxymethylphenyl)-5-oxo-7-thioxo-6-azaspiro[3.4]oct-6-yl]-2-trifluoromethyl-benzonitrile(36b) (0.086 g, 0.2 mmol) and methanesulfonyl anhydride (0.07 g, 0.4mmol) in dichloromethane (1 ml) was stirred at room temperature for 15hours. The mixture was concentrated and chromatographed(dichloromethane:acetone, 98:2) to yield Methanesulfonic acid4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]oct-5-yl]phenylmethylester (50a) [RD154] (0.089 g, 0.175 mmol, 88%), the structure of whichis illustrated in Formula 22, as a white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.63-1.70 (m, 1H), 2.17-2.31 (m, 1H),2.48-2.57 (m, 2H), 2.64-2.70 (m, 2H), 3.04 (s, 3H), 5.30 (s, 2H), 7.37(d, J=8.3 Hz, 2H), 7.62 (d, J=8.3 Hz, 2H), 7.84 (dd, J=8.3, 1.8 Hz, 1H),7.97 (d, J=8.2 Hz, 1H), 7.98 (d, J=1.6 Hz, 1H).

50-2) RD155

Methylamine (0.5 ml) was bubbled into a mixture of Methanesulfonic acid4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]oct-5-yl]phenylmethylester (50a) (0.059 g, 0.115 mmol) in THF (3 ml) cooled to −78° C. After1 hour of reaction at −78° C., the mixture was concentrated andchromatographed (dichloromethane:acetone, 95:5; methanol) to yield4-[5-(4-methylaminomethylphenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]oct-7-yl]-2-trifluoromethylbenzonitrile(50b) [RD155] (0.042 g, 0.095 mmol, 82%), the structure of which isillustrated in Formula 23, as a white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.57-1.70 (m, 1H), 2.16-2.24 (m, 1H), 2.52 (s,3H), 2.53-2.57 (m, 2H), 2.60-2.68 (m, 2H), 3.85 (s, 2H), 7.27 (d, J=8.3Hz, 2H), 7.55 (d, J=8.3 Hz, 2H), 7.84 (dd, J=8.3, 1.8 Hz, 1H), 7.95 (d,J=8.2 Hz, 1H), 7.97 (d, J=1.6 Hz, 1H); ¹³C NMR (CDCl₃, 100 MHz) δ 13.7,31.5, 36.4, 55.6, 67.4, 110.0, 114.9, 122.0 (q, J=272.5 Hz), 127.0 (q,J=4.9 Hz), 129.1, 129.6, 129.8, 132.2, 133.3 (q, J=33.3 Hz), 133.7,135.2, 142.4, 174.8, 179.9.

50-3) RD156

A mixture of Methanesulfonic acid4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]oct-5-yl]phenylmethylester (50a) (0.02 g, 0.039 mmol) and dimethylamine (0.5 ml; distilledfrom its 40% aqueous solution) in THF (1 ml) was stirred for 2 hours at−78° C. The mixture was concentrated and chromatographed(dichloromethane:acetone, 95:5; acetone) to yield4-[5-(4-dimethylaminomethylphenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]oct-7-yl]-2-trifluoromethylbenzonitrile(50c) [RD156] (0.017 g, 0.037 mmol, 95%), the structure of which isillustrated in Formula 24, as a white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.57-1.70 (m, 1H), 2.16-2.24 (m, 1H), 2.32 (s,6H), 2.55-2.60 (m, 2H), 2.63-2.69 (m, 2H), 3.53 (s, 2H), 7.27 (d, J=8.3Hz, 2H), 7.55 (d, J=8.3 Hz, 2H), 7.84 (dd, J=8.3, 1.8 Hz, 1H), 7.95 (d,J=8.2 Hz, 1H), 7.97 (d, J=1.6 Hz, 111); ¹³C NMR (CDCl₃, 100 MHz) δ 13.7,31.5, 45.5, 63.7, 67.4, 110.0, 114.9, 122.0 (q, J=272.5 Hz), 127.0 (q,J=4.9 Hz), 129.1, 129.6, 129.8, 132.2, 133.3 (q, J=33.3 Hz), 133.7,135.2, 142.4, 174.8, 179.9.

Example 51

Sodium cyanide (0.245 g, 5 mmol) was added to a mixture of4-aminobenzoic acid (0.274 g, 2 mmol) and cyclobutanone (0.21 g, 3 mmol)in 90% acetic acid (4.5 ml). The reaction mixture was stirred at roomtemperature for 15 hours. The mixture was washed with aqueous HCl (pH 2)and extracted with ethyl acetate. The organic layer was dried overmagnesium sulfate and concentrated to dryness under vacuum to yield4-(1-cyanocyclobutylamino)benzoic acid (51a) (0.426 g, 1.97 mmol, 99%)as a white solid.

51-1) RD159 and RD160

A mixture of 4-isothiocyanato-2-trifluoromethylbenzonitrile (1a) (0.51g, 2.22 mmol) and 4-(1-cyanocyclobutylamino)benzoic acid (51a) (0.343 g,1.59 mmol) in DMF (2 ml) was heated under microwave irradiation at 60°C. and stirred for 16 hours. To this mixture were added methanol (10 ml)and aq. 2M HCl (5 ml). The second mixture was refluxed for 12 hours.After being cooled to room temperature, the reaction mixture was pouredinto cold water (20 ml) and extracted with ethyl acetate (3×30 ml). Theorganic layer was dried over MgSO₄, concentrated and chromatographed(dichloromethane:acetone, 95:5) to yield4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]oct-5-yl]-benzoicacid methyl ester (51b) [RD159] (0.09 g, 0.196 mmol, 12%), the structureof which is illustrated in Formula 25, as a white powder andN-(3-cyano-4-trifluoromethylphenyl)-4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]oct-5-yl]benzamide(51b′) [RD160] (0.28 g, 0.45 mmol, 29%), the structure of which isillustrated in Formula 26, as a white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.67-1.71 (m, 1H), 2.20-2.26 (m, 1H),2.49-2.57 (m, 2H), 2.66-2.73 (m, 2H), 3.96 (s, 3H), 7.42 (d, J=8.4 Hz,2H), 7.85 (dd, J=8.3, 1.7 Hz, 1H), 7.97 (d, J=8.3 Hz, 1H), 7.98 (d,J=1.7 Hz, 1H), 8.26 (d, J=8.3 Hz, 2H); ¹³C NMR (CDCl₃, 100 MHz) δ 13.7,31.6, 52.6, 67.5, 110.1, 114.8, 121.8 (q, J=272.7 Hz), 127.0 (q, J=4.7Hz), 130.2, 131.4, 131.5, 132.2, 133.4 (q, J=33.2 Hz), 135.2, 137.0,139.2, 165.9, 174.6, 179.7.

¹H NMR (CDCl₃, 400 MHz) δ 1.67-1.71 (m, 1H), 2.18-2.26 (m, 1H),2.50-2.58 (m, 2H), 2.68-2.74 (m, 2H), 7.47 (d, J=8.5 Hz, 2H), 7.83 (d,J=8.7 Hz, 1H), 7.84 (dd, J=8.3, 1.9 Hz, 1H), 7.96 (d, J=8.0 Hz, 1H),9.97 (d, J=1.9 Hz, 1H), 8.10-8.14 (in. 3H). 8.21 (d, J=1.9 Hz, 1H),8.88, (s, 1H).

51-2) RD161

A mixture of4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]oct-5-yl]-benzoicacid methyl ester (51b) (0.046 g, 0.1 mmol) and methylamine (1 mldistilled from its 40% aqueous solution) was kept at −20° C. for 15hours. After evaporation of the methylamine, the mixture waschromatographed (dichloromethane:acetone, 80:20) to yieldN-methyl-4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]oct-5-yl]benzamide(51c) [RD161] (0.041 g, 0.085, 84%), the structure of which isillustrated in Formula 27.

¹H NMR (CDCl₃, 400 MHz) δ 1.63-1.70 (m, 1H), 2.18-2.26 (m, 1H),2.48-2.56 (m, 2H), 2.65-2.71 (tn, 2H), 3.05 (d, J=4.8 Hz, 3H), 6.32 (bs,1H), 7.39 (d, J=8.3 Hz, 2H), 7.84 (dd, J=8.3, 1.7 Hz, 1H), 7.95-7.98 (m,4H); ¹³C NMR (CDCl₃, 100 MHz) δ 13.6, 27.0, 31.6, 67.4, 110.3, 114.8,121.8 (q, J=272.7 Hz), 127.0 (q, J=4.7 Hz), 128.7, 130.3; 132.1, 133.3(q, J=33.2 Hz), 135.2, 136.3, 137.0, 137.8, 167.2, 174.6, 179.8.

Example 52 RD162

Thionyl chloride (2.38 g, 20 mmol) was added slowly to a solution of2-fluoro-4-nitrobenzoic acid (2.97 g. 16 mmol) in DMF (50 ml) cooled at−5° C. The mixture was stirred for an additional 1 hour at −5° C.Methylamine (0.62 g, 20 mmol; freshly distilled from its 40% aqueoussolution) was added to the reaction medium. The second mixture wasstirred for an additional 1 hour. Ethyl acetate (300 ml) was added tothe mixture, which was washed with brine (3×150 ml). The organic layerwas dried over MgSO₄, and concentrated to yieldN-methyl-2-fluoro-4-nitrobenzamide (52a) (2.89 g, 14.6 mmol, 91%) as ayellow solid. ¹H NMR (Acetone d6, 400 MHz) δ 3.05 (d, J=4.3 Hz, 3H),6.31 (dd, J=13.5, 2.1 Hz, 1H), 6.40 (dd, J=8.5, 2.1 Hz, 1H), 7.64 (dd,J=8.6, 8.6 Hz, 1H).

A mixture of N-methyl-2-fluoro-4-nitrobenzamide (52a) (2.89 g, 14.6mmol) and iron (5.04 g, 90 mmol) in ethyl acetate (40 ml) and aceticacid (40 ml) was refluxed for 1 hour. The solid particles were filteredoff. The filtrate was washed with water and extracted with ethylacetate. The organic layer was dried over MgSO₄, concentrated andchromatographed (dichloromethane:acetone, 95:5) to yieldN-methyl-2-fluoro-4-aminobenzamide (52b) (2.3 g, 13.7 mmol, 94%) as anoff-white solid. ¹H NMR (acetone-d₆, 400 MHz) δ 2.86 (d, J=4.3 Hz, 3H),5.50 (bs, 2H), 6.37 (dd, J_(j)=14.7 Hz, J₂=2.1 Hz, 1H), 6.50 (dd, J=8.5,2.1 Hz, 1H), 7.06 (bs, 1H), 7.68 (dd, J=8.8 8.8 Hz, 1H); ¹³C NMR(acetone-d₆, 100 MHz) δ 25.8, 99.6 (d, J=13.8 Hz), 109.2 (d, J=12.8 Hz),110.0 (d, J=1.6 Hz), 132.5 (d, J=4.8 Hz), 153.5 (d, J=12.6 Hz), 162.2(d, J=242.5 Hz), 164.0 (d, J=3.1 Hz).

Sodium cyanide (1.47 g, 30 mmol) was added to a mixture ofN-methyl-2-fluoro-4-aminobenzamide (52b) (1.68 g, 10 mmol) andcyclobutanone (1.4 g, 20 mmol) in 90% acetic acid (20 ml). The reactionmixture was stirred at 80° C. for 24 hours. The mixture was washed withwater and extracted with ethyl acetate. The organic layer was dried overmagnesium sulfate and concentrated to dryness under vacuum. The solidwas washed with a 50:50 mixture of ethyl ether and hexane (10 ml) toremove cyclobutanone cyanohydrin to afford after filtrationN-methyl-4-(1-cyanocyclobutylamino)-2-fluorobenzamide (52c) (2.19 g,8.87 mmol, 89%). ¹H NMR (CDCl₃, 400 MHz) δ 1.87-1.95 (m, 1H), 2.16-2.27(m, 1H), 2.35-2.41 (m, 2H), 2.76-2.83 (m, 2H), 2.97 (d, J=4.4 Hz, 3H),4.68 (bs, 1H), 6.29 (dd, J=14.3, 1.8 Hz, 1H), 6.48 (dd, J=8.3, 1.8 Hz,1H), 6.75 (q, J=4.4 Hz, 1H), 7.90 (dd, J=8.3, 8.3 Hz, 1H); ¹³C NMR(CDCl₃, 100 MHz) δ 15.7, 26.7, 33.9, 49.4, 100.2 (d, J=29.5 Hz), 110.6,111.0 (d, J=11.8 Hz), 133.1 (d, J=4.2 Hz), 148.4 (d, J=12.0 Hz), 162.0(d, J=244.1 Hz), 164.4 (d, J=3.6 Hz).

A mixture of 4-isothiocyanato-2-trifluoromethylbenzonitrile (1a) (2.16g, 9.47 mmol) and N-methyl-4-(1-cyanocyclobutylamino)-2-fluorobenzamide(52c) (1.303 g, 5.27 mmol) in DMF (20 ml) was heated under microwaveirradiation at 80° C. for 16 hours. To this mixture was added methanol(50 ml) and aq. 2N HCl (20 ml). The second mixture was refluxed for 3hours. After being cooled to room temperature, the reaction mixture waspoured into cold water (100 ml) and extracted with ethyl acetate (150ml). The organic layer was dried over MgSO₄, concentrated andchromatographed (dichloromethane:acetone, 95:5) to yieldN-methyl-4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]-2-fluorobenzamide(52d) [RD162] (1.43 g, 3.0 mmol, 57%), the structure of which isillustrated in Formula 28, as a yellow powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.65-1.75 (m, 1H), 2.18-2.30 (m, 1H),2.49-2.57 (m, 2H), 2.67-2.73 (m, 2H), 3.07 (d, J=4.4 Hz, 3H), 6.75 (q,J=4.6 Hz, 1H), 7.17 (dd, J=11.5, 1.9 Hz, 1H), 7.26 (dd, J=8.3, 1.9 Hz,1H), 7.83 (dd, J=8.2, 2.0 Hz, 1H), 7.95 (d, J=1.8 Hz, 1H), 7.97 (d,J=8.3 Hz, 1H) 8.30 (dd, J=8.3, 8.3 Hz, 1H); ¹³C NMR (CDCl₃, 100 MHz) δ13.6, 27.0, 31.7, 67.4, 110.3, 114.8, 118.2, 118.5, 121.9 (q, J=272.7Hz), 126.6, 127.0 (q, J=4.8 Hz), 132.1, 133.3 (q, J=33.2 Hz), 133.8,135.3, 136.8, 139.1 (d, J=10.9 Hz), 160.5 (d, J=249.1 Hz), 162.7 (d,J=3.3 Hz), 174.3, 179.8; ¹⁹F NMR (CDCl₃, 100 MHz) δ 111.13, −62.58.

Example 53 RD163

A mixture of 4-nitro-3-fluorophenol (0.314 g, 2 mmol) and iron (0.56 g,10 mmol) in ethyl acetate (4 ml) and acetic acid (2 ml) was refluxed for3 hour. The solid particles were filtered off. The filtrate was washedwith water and extracted with ethyl acetate. The organic layer was driedover MgSO₄, concentrated to yield 4-amino-3-fluorophenol (53a) (0.25 g,19.6 mmol, 98%) as a brown solid. ¹H NMR (CDCl₃, 400 MHz) δ 6.48-6.58(m, 2H), 6.61-6.70 (m, 1H), 7.87 (bs, 3H).

Sodium cyanide (0.194 g, 4 mmol) was added to a mixture of4-amino-3-fluorophenol (0.29 g, 2.28 mmol) and cyclobutanone (0.175 g,2.5 mmol) in 90% acetic acid (3 ml). The reaction mixture was stirred atroom temperature for 15 hours. The medium was washed with water andextracted with ethyl acetate. The organic layer was dried over magnesiumsulfate, concentrated and chromatographed (dichloromethane:acetone,90:10) to yield1-(2-fluoro-4-hydroxyphenylamino)-cyclobutanecarbonitrile (53b) (0.271g, 1.31 mmol, 58%) as an off-white solid. ¹H NMR (CDCl₃, 400 MHz) δ2.13-2.20 (m, 2H), 2.36-2.41 (m, 2H), 2.70.2.75 (m, 2H), 4.00 (bs, 1H),6.46 (bs, 1H), 6.52 (ddd, J₁=2.2 Hz, J₂=0.65 Hz, J₃=0.22 Hz, 1H), 6.57(d, J=2.3 Hz), 6.62 (dd, J_(f)=3.0 Hz, J₂=0.67 Hz, 1H); ¹³C NMR (CDCl₃,100 MHz) δ 15.7, 34.1, 50.9, 104.0 (d, J=21.9 Hz), 111.0 (d, J=3.4 Hz),115.8 (d, J=3.7 Hz), 121.8, 125.3 (d, J=12.3 Hz), 150.1 (A, J=10.4 Hz),152.8 (d, J=239.3 Hz).

A mixture of 4-isothiocyanato-2-trifluoromethylbenzonitrile (1a) (0.228g, 1.0 mmol) and1-(2-fluoro-4-hydroxyphenylamino)-cyclobutanecarbonitrile (53b) (0.145g, 0.7 mmol) in dry DMF (2 ml) was stirred at room temperature for 24hours. To this mixture were added methanol (10 ml) and aq. 2M HCl (2ml). The second mixture was refluxed for 1 hour. After being cooled toroom temperature, the reaction mixture was poured into cold water (10ml) and extracted with ethyl acetate (50 ml). The organic layer wasdried over MgSO₄, concentrated and chromatographed (dichloromethane pureand then dichloromethane:acetone, 90:10) to yield4-[5-(2-fluoro-4-hydroxyphenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]oct-7-yl]-2-trifluoromethylbenzonitrile(53c) [RD163] (3.17 g, 0.39 mmol, 56%), the structure of which isillustrated in Formula 29, as a off-white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.66-1.75 (m, 1H); 2.18-2.28 (m, 1H),2.42-2.50 (m, 1H), 2.54-2.67 (m, 3H), 6.76 (d, J=2.2 Hz, 2H), 7.15 (t,J=2.1 Hz, 1H), 7.35 (bs, 1H), 7.87 (dd, J_(f)=8.2 Hz, J₂=1.8 Hz, 1H),7.97 (d, J=8.2 Hz, 1H), 7.98 (d, J=1.8 Hz, 1H); ¹³C NMR (CDCl₃, 100 MHz)δ 13.8, 31.0, 67.6, 104.8 (d, J=22.3 Hz), 109.8, 112.6, 114.4 (d, J=13.1Hz), 114.9, 121.9 (q, J=272.8 Hz), 127.1 (q, J=4.8 Hz), 132.0, 132.3,133.5 (q, J=33.3 Hz), 135.3, 137.2, 159.3 (d, J=11.2 Hz), 159.6 (d,J=249.7 Hz), 175.2, 180.5; ¹⁹F NMR (CDCl₃, 100 MHz) δ −117.5, −62.49.

Example 54 RD168

A mixture of 4-nitro-2-fluorobenzonitrile (1.83 g, 5 mmol) and iron(1.68 g, 6 mmol) M a mixture of acetic acid (40 ml) and ethyl acetate(40 ml) was refluxed for 2 hours. The solid was filtered off and thefiltrate was washed with water and extracted with ethyl acetate. Theorganic layer was dried over magnesium sulfate, concentrated andchromatographed (dichloromethane:acetone, 95:5) to yield4-amino-2-fluorobenzonitrile (54a) (0.653 g, 4.8 mmol, 96%).

Sodium cyanide (0.74 g, 15 mmol) was added to a mixture of4-amino-2-fluorobenzonitrile (1.36 g, 10 mmol) and cyclopentanone (1.26g, 15 mmol) in 90% acetic acid (10 ml). The reaction mixture was stirredat room temperature for 3 hours and then the medium was hearted to 80°C. and stirred for an additional 5 hours. The medium was washed withwater and extracted with ethyl acetate. The organic layer was dried overmagnesium sulfate, concentrated and chromatographed(dichloromethane:acetone, 97:3) to yield4-(1-cyanocyclopentylamino)-2-fluorobenzonitrile (54b) (2.07 g, 9.03mmol, 90%) as a yellow solid. ¹H NMR (CDCl₃, 400 MHz) δ 1.69-1.91 (m,4H), 2.13-2.18 (m, 2H), 2.37-2.42 (m, 2H), 5.08 (bs, 1H), 6.54-6.62 (m,2H), 7.39 (t, J=7.3 Hz, 1H); ¹³C NMR (CDCl₃, 100 MHz) δ 23.7, 39.8,56.8, 89.6 (d, J=15.8 Hz), 101.2 (d, J=23.8 Hz), 110.9, 115.2, 120.8,134.1 (d, J=2.4 Hz), 150.3 (d, J=11.2 Hz), 164.5 (d, J=254.1 Hz).

A mixture of 4-isothiocyanato-2-trifluoromethylbenzonitrile (1a) (0.171g, 0.75 mmol) and 4-(1-cyanocyclopentylamino)-2-fluorobenzonitrile (54b)(0.115 g, 0.5 mmol) in dry DMF (1 ml) was heated under microwaveirradiation at 60° C. for 48 hours. To this mixture were added methanol(3 ml) and aq 2M HCl (2 ml). The second mixture was refluxed for 1 hour.After being cooled to room temperature, the reaction mixture was pouredinto cold water (10 ml) and extracted with ethyl acetate (15 ml). Theorganic layer was dried over MgSO₄, concentrated and chromatographed(dichloromethane:acetone, 98:2) to yield4-[1-(4-cyano-3-fluorophenyl)-4-oxo-2-thioxo-1,3-diazaspiro[4.4]non-3-yl]-2-trifluoromethylbenzonitrile(54c) [RD168] (0.017 g, 0.037 mmol, 7%), of which the structure isillustrated in Formula 30, as an off-white powder.

¹H NMR (CDCl₃, 400 MHz) δ 1.53-1.63 (m, 2H), 1.89-2.00 (m, 2H),2.09-2.16 (m, 2H), 2.35-2.42 (m, 2H), 7.27-7.37 (m, 2H), 7.78-7.90 (m,3H), 7.95 (d, J=1.8 Hz, 1H), 7.97 (d, J=8.3 Hz, 1H); ¹³C NMR (CDCl₃, 100MHz) δ 25.2, 36.5, 75.3, 103.2 (d, J=15.3 Hz), 110.4, 112.8, 114.7,119.2 (d, J=20.7 Hz), 121.9 (q, J=272.8 Hz), 127.0 (q, J=4.8 Hz), 132.1,133.7 (q, J=33.2 Hz), 134.6, 135.3, 135.8, 136.8, 141.8 (d, J=9.5 Hz),163.4 (d, J=261.5 Hz), 175.3, 180.1.

Example 55 RD136 and RD142

Additional diarylhydantoin compounds can be synthesized, including thefollowing compounds illustrated in Formulas 35 and 36.

Example 56 RD162′

In the following, air or moisture sensitive reactions were conductedunder argon atmosphere using oven-dried glassware and standardsyringe/septa techniques. The reactions were monitored with a SiO₂ TLCplate under UV light (254 nm) followed by visualization with ap-anisaldehyde or ninhydrin staining solution. Column chromatography wasperformed on silica gel 60. ¹H NMR spectra were measured at 400 MHz inCDCl₃ unless stated otherwise and data were reported as follows in ppm(δ) from the internal standard (TMS, 0.0 ppm): chemical shift(multiplicity, integration, coupling constant in Hz.).

Periodic acid (1.69 g, 7.41 mmol) was dissolved in acetonitrile (25 mL)by vigorous stirring, and then chromium trioxide (0.16 g, 1.60 mmol) wasdissolved into the solution. 2-Fluoro-4-nitrotoluene (0.33 g, 2.13 mmol)was added to the above solution with stirring. A white precipitateformed immediately with exothermic reaction. After 1 h of stirring, thesupernatant liquid of the reaction mixture was decanted to a flask, andthe solvent was removed by evaporation. The residues were extracted withmethylene chloride (2×30 mL) and water (2×30 mL). The organic layer wasdried over MgSO₄, and concentrated to give 2-Fluoro-4-nitrobenzoic acid(Formula 37) (0.32 mg, 81%) as a white solid. ¹H NMR δ 8.06 (ddd, 1H,J=9.9, 2.2 and 0.3), 8.13 (ddd, 1H, J=8.6, 2.2 and 0.9), 8.25 (ddd, 1H,J=8.6, 7.0 and 0.3).

Thionyl chloride (0.15 g, 1.30 mmol) was added slowly to a solution of2-fluoro-4-nitrobenzoic acid (Formula 37) (0.20 g, 1.10 mmol) in DMF (5mL) cooled at −5° C. The mixture was stirred for an additional 1 hour at−5° C. Excess methylamine (freshly distilled from its 40% aqueoussolution) was added to the reaction medium. The second mixture wasstirred for an additional 1 hour. Ethyl acetate (50 mL) was added to themixture, which was washed with brine (2×50 ml). The organic layer wasdried over MgSO₄, and concentrated to yieldN-Methyl-2-fluoro-4-nitrobenzamide (Formula 38) (0.18 g, 85%) as ayellowish solid. ¹H NMR (acetone-d₆) δ 3.05 (d, 3H, J=4.3), 6.31 (dd,1H, J=13.5 and 2.1), 6.40 (dd, 1H, J=8.6 and 2.1), 7.64 (dd, 1H, J=8.6and 8.6).

A mixture of N-Methyl-2-fluoro-4-nitrobenzamide (Formula 38) (0.18 g,0.91 mmol) and iron (0.31 g, 5.60 mmol) in ethyl acetate (5 mL) andacetic acid (5 mL) was refluxed for 1 h. The solid particles werefiltered off. The filtrate was washed with water and extracted withethyl acetate. The organic layer was dried over MgSO₄, concentrated andthe residue was purified with SiO₂ column chromatography(dichloromethane:acetone, 95:5) to giveN-Methyl-2-fluoro-4-aminobenzamide (Formula 39) (0.14 g, 92%) as anoff-white solid. ¹H NMR (acetone-d₆) δ 2.86 (d, 3H, J=4.3), 5.50 (br s,2H), 6.37 (dd, 1H, J=14.7 and 2.1), 6.50 (dd, 1H, J=8.6 and 2.1), 7.06(br s, 1H), 7.68 (dd, 111, J=8.8 and 8.8).

A mixture of N-Methyl-2-fluoro-4-aminobenzamide (Formula 39) (96 mg,0.57 mmol), acetone cyanohydrin (0.3 mL, 3.14 mmol) and magnesiumsulfate (50 mg) was heated to 80° C. and stirred for 12 h. To the mediumwas added ethyl acetate (25 mL) and then washed with water (2×25 mL).The organic layer was dried over MgSO₄ and concentrated and the residuewas purified with SiO₂ column chromatography (dichloromethane:acetone,95:5) to giveN-Methyl-2-fluoro-4-(1,1-dimethyl-cyanomethyl)-aminobenzamide (Formula40) (101 mg, 75%) as a white solid. ¹H NMR δ 1.74 (s, 6H), 2.98 (dd, 3H,J=4.8 and 1.1), 6.58 (dd, 1H, J=14.6 and 2.3), 6.63 (dd, 1H, J=8.7 and2.3), 6.66 (br s, 1 H), 7.94 (dd, 1H, J=8.7 and 8.7).

4-Amino-2-trifluoromethylbenzonitrile (2.23 g, 12 mmol) was addedportionwise over 15 min into a well-stirred heterogeneous mixture ofthiophosgene (1 mL, 13 mmol) in water (22 mL) at room temperature.Stirring was continued for an additional 1 h. The reaction medium wasextracted with chloroform (3×15 ml). The combined organic phase wasdried over MgSO₄ and evaporated to dryness under reduced pressure toyield desired product 4-Isothiocyanato-2-trifluoromethylbenzonitrile(Formula 41) as brownish solid and was used as such for the next step(2.72 g, 11.9 mmol, 99%). ¹H NMR δ 7.49 (dd, 1H, J=8.3 and 2.1), 7.59(d, 1H, J=2.1), 7.84 (d, 1H, J=8.3).

56-1) RD162′

A mixture ofN-Methyl-2-fluoro-4-(1,1-dimethyl-cyanomethyl)-aminobenzamide (Formula40) (30 mg, 0.13 mmol) and4-Isothiocyanato-2-trifluoromethylbenzonitrile (Formula 41) (58 mg, 0.26mmol) in DMF (1 mL) was heated under microwave irradiation at 100° C.for 11 hours. To this mixture was added methanol (20 mL) and aq. 1N HCl(5 mL). The second mixture was refluxed for 1.5 h. After being cooled toroom temperature, the reaction mixture was poured into cold water (50mL) and extracted with ethyl acetate (50 mL). The organic layer wasdried over MgSO₄, concentrated and the residue was purified with SiO₂column chromatography (dichloromethane:acetone, 95:5) to give RD162′(Formula 42) (15 mg, 25%) as a colorless crystal. ¹H NMR & 1.61 (s, 6H),3.07 (d, 3H, J=4.1), 6.71 (m, 1H), 7.15 (dd, 11-1, J=11.7 and 2.0), 7.24(dd, 1H, J=8.4 and 2.0), 7.83 (dd, 1H, J=8.2 and 2.1), 7.95 (d, 1H,J=2.1), 7.99 (d, 1H, J=8.2), 8.28 (dd, 1H, J=8.4 and 8.4).

Example 57

A mixture of N-Methyl-2-fluoro-4-aminobenzamide (Formula 39) (62 mg,0.37 mmol), cyclopentanone (0.07 mL, 0.74 mmol) and TMSCN (0.1 mL, 0.74mmol) was heated to 80° C. and stirred for 13 h. To the medium was addedethyl acetate (2×20 mL) and then washed with water (2×20 mL). Theorganic layer was dried over MgSO₄ and concentrated and the residue waspurified with silica gel column chromatography (dichloromethane:acetone,95:5) to give N-Methyl 2-fluoro-4-(1-cyanocyclopentyl)aminobenzamide(Formula 43) (61 mg, 63%) as a white solid. ¹H NMR δ 7.95 (dd, 1H,J=8.8, 8.8 Hz), 6.65 (br s, 1H), 6.59 (dd, 1H, J=8.8, 2.3 Hz), 6.50 (dd,1H, J=14.6, 2.3 Hz), 4.60 (br s, 1H), 2.99 (dd, 3H, J=4.8, 1.1 Hz),2.36-2.45 (m, 2H), 2.10-2.18 (m, 2H), 1.82-1.95 (m, 4H).

57-1) RD162″

A mixture of N-Methyl 2-fluoro-4-(1-cyanocyclopentyl)aminobenzamide(Formula 43) (57 mg, 0.22 mmol) and 4-isothiocyanato-2-trifluoromethylbenzonitrile (0.15 g, 0.65 mmol) in DMF (3 mL) was heated undermicrowave irradiation (open vessel) at 130° C. for 12 hours. To thismixture was added methanol (20 mL) and aq. 1 N HCl (5 mL). The secondmixture was refluxed for 1.5 h. After being cooled to room temperature,the reaction mixture was poured into cold water (50 mL) and extractedwith ethyl acetate (50 mL). The organic layer was dried over MgSO₄,concentrated and the residue was purified with silica gel columnchromatography (dichloromethane:acetone, 95:5) to give4-(3-(4-Cyano-3-(trifluoromethyl)phenyl)-4-oxo-2-thioxo-1,3-diazaspiro[4.4]nonan-1-yl)-2-fluoro-N-methylbenzamide,RD162″ (Formula 44) (8 mg, 7%) as a pale yellowish solid. ¹H NMR δ 8.28(dd, 1H, J=8.4, 8.4 Hz), 7.98 (d, 1H, J=8.3 Hz), 7.96 (d, 1H, J=1.8 Hz),7.84 (dd, 1H, J=8.3, 1.8 Hz), 7.27 (dd, 1H, J=8.4, 1.8 Hz), 7.17 (dd,1H, J=11.7, 1.8 Hz), 6.67-6.77 (m, 1H), 3.07 (d, 3H, J=4.3 Hz),2.32-2.41 (m, 2H), 2.13-2.21 (m, 2H), 1.85-1.96 (m, 2H), 1.49-1.59 (m,2H).

Example 58

Trifluoroacetic anhydride (0.85 mL, 6.14 mmol) was added to a solutionof 4-(4-aminophenyl)butyric acid (0.5 g, 2.79 mmol) in chloroform (10mL) at 0° C. The mixture was warmed to room temperature and stirred for3 hours. The mixture was partitioned with chloroform (20 mL) and water(20 mL). The organic layer was dried over MgSO₄, concentrated and theresidue was purified with silica gel column chromatography(dichloromethane:acetone, 9:1) to give4-[4-(2,2,2-Trifluoroacetylamino)phenyl]butanoic acid (Formula 45) (0.53g, 69%). ¹H NMR δ 7.81 (br s, 1H), 7.48 (d, 2H, J=8.5 Hz), 7.22 (d, 2H,J=8.5 Hz), 2.68 (t, 2H, J=7.5 Hz), 2.38 (t, 211, J=7.5 Hz), 1.96 (p, 2H,J=7.5 Hz).

Thionyl chloride (71 mg, 0.60 mmol) was added slowly to a solution of4-[4-(2,2,2-Trifluoroacetylamino)phenyl]butanoic acid (Formula 45) (0.15g, 0.55 mmol) in DMF (5 mL) cooled at −5° C. The mixture was stirred foran additional 1 hour at −5° C. Excess dimethylamine (freshly distilledfrom its 40% aqueous solution) was added to the reaction medium. Thesecond mixture was stirred for an additional 1 hour. Ethyl acetate (50mL) was added to the mixture, which was washed with brine (2×50 ml). Theorganic layer was dried over MgSO₄, and concentrated to yieldN,N-Dimethyl 4-[4-(2,2,2-Trifluoroacetylamino)phenyl]butanamide (Formula46) (0.17 g, quant.) as a yellowish solid. ¹H NMR 9.70 (br s, 1H), 7.55(d, 2H, J=8.6 Hz), 7.11 (d, 2H, J=8.6 Hz), 2.91 (s, 3H), 2.89 (s, 3H),2.60 (t, 2H, J=7.7 Hz), 2.27 (t, 2H, J=7.7 Hz), 1.89 (p, 2H, J=7.7 Hz).

1 N NaOH solution (3 mL) was added to a solution of N,N-Dimethyl4-[4-(2,2,2-Trifluoroacetylamino)phenyl]butanamide (Formula 46) (0.17 g,0.55 mmol) in methanol (2 mL) at room temperature. The mixture wasstirred for 14 hour. The mixture was partitioned with chloroform (25 mL)and water (25 mL). The organic layer was dried over MgSO₄, andconcentrated and the residue was purified with silica gel columnchromatography (dichloromethane:acetone, 9:1) to give N,N-Dimethyl4-(4-aminophenyl)butanamide (Formula 47) (74 mg, 66%) as a white solid.¹H NMR δ 6.97 (d, 2H, J=8.3 Hz), 6.61 (d, 2H, J=8.3 Hz), 3.56 (br s,2H), 2.92 (s, 6H), 2.56 (t, 2H, J=7.7 Hz), 2.28 (t, 2H, J=7.7 Hz), 1.91(p, 2H, J=7.7 Hz).

A mixture of N,N-Dimethyl 4-(4-aminophenyl)butanamide (Formula 47) (74mg, 0.36 mmol), cyclobutanone (54 mg, 0.78 mmol) and TMSCN (77 mg, 0.78mmol) was heated to 80° C. and stirred for 15 h. To the medium was addedethyl acetate (2×20 mL) and then washed with water (2×20 mL). Theorganic layer was dried over MgSO₄ and concentrated and the residue waspurified with silica gel column chromatography (dichloromethane:acetone,9:1) to give N,N-Dimethyl 4-[4-(1-cyanocyclobutylamino)phenyl]butanamide(Formula 48) (58 mg, 57%) as a white solid. ¹H NMR δ 7.07 (d, 2H, J=8.5Hz), 6.59 (d, 2H, J=8.5 Hz), 3.94 (br s, 1H), 2.94 (s, 3H), 2.93 (s,3H), 2.75-2.83 (m, 2H), 2.60 (t, 2H, J=7.6 Hz), 2.33-2.42 (m, 2H), 2.30(t, 2H, J=7.6 Hz), 2.11-2.28 (m, 2H), 1.93 (p, 2H, J=7.6 Hz).

A mixture of N,N-Dimethyl 4-[4-(1-cyanocyclobutylamino)phenyl]butanamide(Formula 48) (58 mg, 0.20 mmol) and 4-isothiocyanato-2-trifluoromethylbenzonitrile (74 mg, 0.32 mmol) in DMF (3 mL) was heated under refluxfor 2 hours. To this mixture was added methanol (20 mL) and aq. 1 N HCl(5 mL). The second mixture was refluxed for 1.5 h. After being cooled toroom temperature, the reaction mixture was poured into cold water (50mL) and extracted with ethyl acetate (50 mL). The organic layer wasdried over MgSO₄, concentrated and the residue was purified with silicagel column chromatography (dichloromethane:acetone, 95:5) to give4-(4-(7-(4-Cyano-3-(trifluoromethyl)phenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)phenyl)-N,N-dimethylbutanamide,RD169 (Formula 49) (44 mg, 42%) as a pale yellowish solid. ¹H NMR δ 7.98(s, 1H), 7.97 (d, 1H, J=8.2 Hz), 7.86 (d, 1H, J=8.2 Hz), 7.42 (d, 2H,J=8.3 Hz), 7.22 (d, 2H, J=8.3 Hz), 2.99 (s, 3H), 2.96 (s, 3H), 2.78 (t,2H, J=7.5 Hz), 2.62-2.70 (m, 2H), 2.52-2.63 (m, 2H), 2.40 (t, 2H, J=7.5Hz), 2.15-2.30 (m, 1H), 2.04 (p, 2H, J=7.5 Hz), 1.62-1.73 (m, 1H).

Example 59

A mixture of 4-(4-aminophenyl)butyric acid (0.20 g, 1.12 mmol),cyclobutanone (0.17 mL, 2.23 mmol) and TMSCN (0.30 mL, 2.23 mmol) washeated to 80° C. and stirred for 13 h. To the medium was added ethylacetate (2×30 mL) and then washed with water (2×30 mL). The organiclayer was dried over MgSO₄ and concentrated and the residue was purifiedwith silica gel column chromatography (dichloromethane:acetone, 9:1) togive 4-[4-(1-Cyanocyclobutylamino)phenyl]butanoic acid (Formula 50)(0.21 g, 74%) as a yellowish solid. ¹H NMR δ 7.06 (d, 2H, J=8.6 Hz),6.59 (d, 2H, J=8.6 Hz), 2.75-2.83 (m, 2H), 2.59 (t, 2H, J=7.5 Hz), 2.37(t, 2H, J=7.5 Hz), 2.33-2.42 (m, 2H), 2.11-2.28 (m, 2H), 1.92 (p, 2H,J=7.5 Hz).

A mixture of 4-[4-(1-Cyanocyclobutylamino)phenyl]butanoic acid (Formula50) (0.21 g, 0.83 mmol) and 4-isothiocyanato-2-trifluoro benzonitrile(0.25 g, 1.08 mmol) in toluene (10 mL) was heated under reflux for 1hours. To this mixture was added aq. 1 N HCl (5 mL). The second mixturewas refluxed for 1.5 h. After being cooled to room temperature, thereaction mixture was poured into cold water (50 mL) and extracted withethyl acetate (50 mL). The organic layer was dried over MgSO₄,concentrated and the residue was purified with silica get columnchromatography (dichloromethane:acetone, 95:5) to give4-(4-(7-(4-Cyano-3-(trifluoromethyl)phenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)phenyl)butanoicacid, RD141 (Formula 51) (60 mg, 15%). ¹H NMR δ 7.98 (d, 1H, J=1.8 Hz),7.97 (d, 1H, J=8.3 Hz), 7.86 (dd, 1H, J=8.3, 1.8 Hz), 7.42 (d, 2H, J=8.5Hz), 7.24 (d, 2H, J=8.5 Hz), 2.79 (t, 2H, J=7.5 Hz), 2.62-2.68 (m, 2H),2.51-2.59 (m, 2H), 2.47 (t, 2H, J=7.5 Hz), 2.14-2.26 (m, 1H), 2.06 (p,2H, J=7.5 Hz), 1.60-1.70 (m, 1H).

Example 60

To a solution of4-(4-(7-(4-Cyano-3-(trifluoromethyl)phenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)phenyl)butanoicacid, RD141 (Formula 51) (60 mg, 0.12 mmol) in DMF (3 mL) was addedthionyl chloride (0.01 mL, 0.15 mmol) at 0° C. The mixture was stirredat 0° C. for 1 hour. Then ammonia was bubbled into the mixture. Themixture was partitioned with ethyl acetate (25 mL) and water (25 mL).The organic layer was dried over MgSO₄, concentrated and chromatographed(dichloromethane:acetone, 70:30) to yield4-(4-(7-(4-Cyano-3-(trifluoromethyl)phenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)phenyl)butanamide,RD130 (Formula 52) (37 mg, 61%) as a white powder. ¹H NMR δ 7.97 (d, 1H,J=1.8 Hz), 7.95 (d, 1H, J=8.3 Hz), 7.85 (dd, 1H, J=8.3 Hz), 7.39 (d, 2H,J=8.3 Hz), 7.22 (d, 211, J=8.3 Hz), 5.59 (br s, 2H), 2.77 (t, 21-1,J=7.5 Hz), 2.62-2.68 (m, 2H), 2.51-2.59 (m, 2H), 2.31 (t, 2H, J=7.5 Hz),2.16-2.25 (m, 1H), 2.05 (p, 2H, J=7.5 Hz), 1.57-1.70 (m, 1H).

Example 61

A solution of DMSO (0.01 mL, 0.12 mmol) in dry dichloromethane (1 mL)was added to a stirred solution of oxalyl chloride (0.01 mL, 0.09 mmol)in dry dichloromethane (2 mL) at −78° C. After 15 min, a dichloromethanesolution of4-(4-(7-(4-Cyano-3-(trifluoromethyl)phenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)phenyl)butanamide,RD130 (Formula 52) (35 mg, 0.07 mmol) was added to the reaction mixture.Stirring was continued for 20 min at −78° C., and then biethylamine(0.03 mL, 0.22 mmol) was added. After 30 min at −78° C., the reactionmixture was warmed to room temperature and then reaction was quenchedwith saturated aq. NH₄C1 solution. The reaction mixture was diluted withdichloromethane, and extracted with dichloromethane. The organic layerwas dried over MgSO₄, concentrated and chromatographed(dichloromethane:acetone, 95:5) to yield4-(5-(4-(3-Cyanopropyl)phenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-2-(trifluoromethyl)benzonitrile,RD170 (Formula 53) (29 mg, 87%) as a viscous oil. ¹H NMR δ 7.98 (d, 1H,J=1.8 Hz), 7.98 (d, 1H, J=8.3 Hz), 7.86 (dd, 1H, J=8.3, 1.8 Hz), 7.43(d, 2H, J=8.4 Hz), 7.27 (d, 2H, J=8.4 Hz), 2.90 (t, 2H, J=7.3 Hz),2.63-2.73 (m, 2H), 2.52-2.62 (m, 2H), 2.42 (t, 2H, J=7.3 Hz), 2.18-2.30(m, 1H), 2.07 (p, 2H, J=7.3 Hz), 1.63-1.73 (m, 1H).

One skilled in the art could modify and/or combine the synthesesdescribed herein to make other diarylhydantoin compounds.

Inventive compounds also include those with the following formulas.

Where R is selected from hydrogen, aryl, substituted aryl, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, halogenated alkyl, halogenated alkenyl, halogenated akynyl,arylalkyl, arylalkenyl, arylalkynyl, heterocyclic aromatic ornon-aromatic, substituted heterocyclic aromatic or non-aromatic,cycloalkyl, substituted cycloalkyl, halogen, SO₂R₁₁, NR₁₁R₁₂,NR₁₂(CO)OR₁₁, NH(CO)NR₁₁R₁₂, NR₁₂(CO)R₁₁, O(CO)R₁₁, O(CO)OR₁₁, O(CS)R₁₁,NR₁₂(CS)R₁₁, NH(CS)NR₁₁R₁₂, NR₁₂(CS)OR₁₁.

R₁ and R₂ are independently selected from hydrogen, aryl, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, halogenated alkyl, halogenated alkenyl, halogenated akynyl,arylalkyl, arylalkenyl, arylallynyl, heterocylic aromaric ornon-aromatic, substituted heterocyclic aromatic or non-aromatic,cycloalkyl, substituted cycloalkyl.

R₁ and R₂ can be connected to form a cycle which can be heterocyclic,substituted heterocyclic, cycloakyl, substituted cycloalkyl.

R₃ is selected from aryl, substituted aryl, alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, arylalkyl,arylalkenyl, arylalkynyl, heterocyclic aromatic or non-aromatic,substituted heterocyclic aromatic or non-aromatic; cycloalkyl;substituted cycloalkyl, SO₂R₁₁, NR₁₁R₁₂, (CO)OR₁₁, (CO)NR₁₁R₁₂, (CO)R₁₁,(CS)R₁₁, (CS)R₁₁, (CS)NR₁₁R₁₂, (CS)OR₁₁.

R₅ is CN or NO₂ or SO₂R₁₁

R₆ is CF₃, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, halogenated alkyl, halogenated alkenyl,halogenated akynyl, halogen.

A is sulfur atom (S) or oxygen atom (O).

B is O or S or NR₃

X is carbon or nitrogen and can be at any position in the ring.

R₁₁ and R₁₂ are independently selected from hydrogen, aryl, aralkyl,substituted aralkyl, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, halogenated alkyl, halogenatedalkenyl, halogenated akynyl, arylalkyl, arylalkenyl, arylalkynyl,heterocyclic aromatic or non-aromatic, substituted heterocyclic aromaticor non-aromatic, cyclolakyl, substituted cycloalkyl.

R₁₁ and R₁₂ can be connected to form a cycle which can be heterocyclicaromatic or non-aromatic, substituted heterocyclic aromatic, cycloakyl,substituted cycloalkyl.

Pharmacological Examination of the Compounds

Compounds for which synthetic routes are described above were identifiedthrough screening on hormone refractory prostate cancer cells forantagonistic and agonistic activities against AR utilizing screeningprocedures similar to those in PCT applications US04/42221 andUS05/05529, which are hereby incorporated by reference. A number ofcompounds exhibited potent antagonistic activities with minimalagonistic activities for over expressed AR in hormone refractoryprostate cancer.

In Vitro Biological Assay Effect of Compounds on AR by a Reporter Assay

The compounds were subjected to tests using an artificial AR responsereporter system in a hormone refractory prostate cancer cell line. Inthis system, the prostate cancer LNCaP cells were engineered to stablyexpress about 5-fold higher level of AR than endogenous level. Theexogenous AR has similar properties to endogenous AR in that both arestabilized by a synthetic androgen R1881. The AR-over expressed cellswere also engineered to stably incorporate an AR response reporter andthe reporter activity of these cells shows features of hormonerefractory prostate cancer. It responds to low concentration of asynthetic androgen R1881, is inhibited only by high concentrations ofbicalutamide (see Table 1), and displays agonistic activity withbicalutamide (FIG. 1 and Table 2). Consistent with published data,bicalutamide inhibited AR response reporter and did not have agonisticactivity in hormone sensitive prostate cancer cells (FIG. 2).

We examined the antagonistic activity of the compounds for which thesynthesis is described above in the presence of 100 pM of R1881.Engineered LNCaP cells (LNCaP-AR, also abbreviated LN-AR) weremaintained in Iscove's medium containing 10% fetal bovine serum (FBS).Two days prior to drug treatment, the cells were grown in Iscove'smedium containing 10% charcoal-stripped FBS (CS-FBS) to deprive ofandrogens. The cells were split and grown in Iscove's containing 10%CS-FBS with 100 pM of R1881 and increasing concentrations of testcompounds. After two days of incubation, reporter activities wereassayed.

Table 1 lists the IC50 of these compounds to inhibit AR in hormonerefractory prostate cancer. The control substance bicalutamide has an1050 of 889 nM. Most of the compounds identified (diarylthiohydantoirts)have IC50s between 100 to 200 nM in inhibiting AR in hormone refractoryprostate cancer. In contrast, antiandrogenic compounds listed asexamples in U.S. Pat. No. 5,705,654, such as examples 30-2, 30-3, 31-2,31-3, and 24-3 (RD73-RD77) have no inhibitory activities on AR in thissystem.

TABLE 1 Antagonistic activities against AR in hormone refractoryprostate cancer, measured by an AR response reporter and by endogenousPSA expression. IC50 (nM) IC50 (nM) Example Name Reporter PSABicalutamide N-[4-cyano-3-(trifluoromethyl)phenyl]-3-[(4- 889 >1000Comparative fluorophenyl)sulfonyl]-2-hydroxy-2- methylpropanamide 294-[3-(4-hydroxybutyl)-4,4-dimethyl-5-oxo-2- No(*) No Comparativethioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile 6-24-[3-phenyl-4,4-dimethyl-5-oxo-2-thioxoimidazolidin- 149 n/a(**) (6b)1-yl]-2-trifluoromethylbenzonitrile [RD10] 5-3b4-[3-(4-methylphenyl)-4,4-dimethyl-5-oxo-2- 125 132 (5c)thioxoimidazolidin-1-yl]-2-trifluoromethyl- [RD7] benzonitrile 3-34-[3-(4-hydroxyphenyl)-4,4-dimethyl-5-oxo-2- 137 122 (3c) [RD8]thioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile 2-44-[3-(4-aminophenyl)-4,4-dimethyl-5-oxo-2- 273 n/a (2d) [RD9]thioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile 4 Chloroaceticacid 4-[3-(4-cyano-3- 131 n/a (4a) [RD13]trifluoromethylphenyl)-5,5-dimethyl-4-oxo-2-thioxoimidazolidin-1-yl]phenyl ester 8-24-(4-Oxo-2-thioxo-1-(4-methylphenyl)-1,3- 147 n/a (8b) [RD35]diazaspiro[4.4]non-3-yl)-2-trifluoromethylbenzonitrile 7-3b4-(8-oxo-6-thioxo-5-(4-methylphenyl)-5,7- 124 128 (7c) [RD37]diazaspiro[3.4]oct-7-yl)-2-trifluoromethylbenzonitrile 9-34-(4-Oxo-2-thioxo-1-(4-methylphenyl)-1,3- 194 n/a (9c) [RD48]diazaspiro[4.5]dec-3-yl)-2-trifluoromethylbenzonitrile 10-34-(4-oxo-2-thioxo-1-(4-methylphenyl)-1,3- 232 n/a (10c) [RD49]diazaspiro[4.5]undec-3-yl)-2- trifluoromethylbenzonitrile 284-(8-methyl-4-oxo-2-thioxo-1,3,8-triazaspiro[4.5]dec- No n/a Comparative3-yl)-2-trifluoromethylbenzonitrile (28a) [RD52] 27-34-(8-methyl-4-oxo-2-thioxo-1-(4-methylphenyl)-1,3,8- 638 n/a (27c)[RD53] triazaspiro[4.5]dec-3-yl)-2-trifluoromethylbenzonitrile 264-[1-(4-cyanophenyl)-4-oxo-2-thioxo-1,3- 469 n/a (26a) [RD54]diazaspiro[4.4]non-3-yl]-2-trifluoromethylbenzonitrile 254-[1-(4-nitrophenyl)-4-oxo-2-thioxo-1,3- 498 n/a (25a) [RD55]diazaspiro[4.4]non-3-yl]-2-trifluoromethylbenzonitrile 12-24-(8-oxo-6-thioxo-5-(4-biphenyl)-5,7- 283 n/a (12b) [RD57]diazaspiro[3.4]oct-7-yl)-2-trifluoromethylbenzonitrile 11-24-(8-oxo-6-thioxo-5-(4-hydroxyphenyl)-5,7- 162 n/a (11b) [RD58]diazaspiro[3.4]oct-7-yl)-2-trifluoromethylbenzonitrile 174-[3-(4-hydroxyphenyl)-4,4-dimethyl-2,5- 278 287 (17a) [RD59]dithioxoimidazolidin-1-yl]-2- trifluoromethylbenzonitrile 184-[3-(4-hydroxyphenyl)-4,4-dimethyl-2,5- 369 511 (18a) [RD60]dioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile 22-22-[3-(4-cyano-3-trifluoromethylphenyl)-5,5-dimethyl- 523 >500 (22b)[RD65] 4-oxo-2-thioxoimidazolidin-1-yl]benzoic acid 20-24-(4,4-dimethyl-5-oxo-2-thioxo-3-(4- 143 144 (20b) [RD66]trifluoromethylphenyl)imidazolidin-1-yl)-2- trifluoromethylbenzonitrile21-2 4-(4,4-bischloromethyl-5-oxo-2-thioxo-3-(4- 521 >500 (21b) [RD67]methylphenyl)imidazolidin-1-yl)-2- trifluoromethylbenzonitrile 19-24-(4-fluoromethyl-4-methyl-5-oxo-2-thioxo-3-(4- 126 129 (19b) [RD68]methylphenyl)imidazolidin-1-yl)-2- trifluoromethylbenzonitrile 23-24-(8-oxo-6-thioxo-5-(2-methylphenyl)-5,7- 258 232 (23b) [RD71]diazaspiro[3.4]oct-7-yl)-2-trifluoromethylbenzonitrile 30-24-(5-methyl-8-oxo-6-thioxo-5,7-diazaspiro[3.4]oct-7- No No Comparativeyl)-2-trifluoromethylenzonitrile (30b) [RD73] 30-34-(5-methyl-6,8-dioxo-5,7-diazaspiro[3.4]oct-7-yl)-2- No No Comparativetrifluoromethylbenzonitrile (30c) [RD74] 31-24-(1-methyl-4-oxo-2-thioxo-1,3-diazaspiro[4.4]non-3- No No Comparativeyl)-2-trifluoromethylbenzonitrile (31b) [RD75] 31-34-(1-methyl-2,4-dioxo-1,3-diaza-spiro[4.4]non-3-yl)- No No Comparative2-trifluoromethylbenzonitrile (31c) [RD76] 24-34-(4-oxo-2-thioxo-1,3-diazaspiro[4.4]non-3-yl)-2- No No Comparativetrifluoromethylbenzonitrile (24c) [RD77] 15-24-[4,4-dimethyl-3-(4-pyridin-2-yl)-5-oxo-2- 723 n/a (15b) [RD82]thioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile 14-24-[4,4-dimethyl-3-(4-methylpyridin-2-yl)-5-oxo-2- 457 n/a (14b) [RD83]thioxoimidazolidin-1-yl]-2-trifluoromethylbenzonitrile 16-24-[5-(5-methyl-2H-pyrazol-3-yl)-8-oxo-6-thioxo-5,7- >1000 n/aComparative diaza-spiro[3.4]oct-7-yl]-2-trifluoromethyl- (16b) [RD84]benzonitrile 13-2 4-(8-oxo-6-thioxo-5-(4-biphenyl)-5,7- >1000 n/a (12b)[RD85] diazaspiro[3.4]oct-7-yl)-2-trifluoromethylbenzonitrile 324-(8-methylimino-6-thioxo-5-p-tolyl-5,7-diaza- 222 421 (32a) [RD90]spiro[3.4]oct-7-yl)-2-trifluoromethyl-benzonitrile 331-[3-(4-cyano-3-trifluoromethyl-phenyl)-5,5-dimethyl- 157 239 (33a)[RD91] 2-thioxo-1-p-tolyl-imidazolidin-4-ylidene]-3-ethyl- thiourea 341-[7-(4-cyano-3-trifluoromethyl-phenyl)-6-thioxo-5-p- 176 276 (34a)[RD92] tolyl-5,7-diaza-spiro[3.4]oct-8-ylidene]-3-phenyl- thiourea 351-(4-Cyano-3-trifluoromethyl-phenyl)-3-[7-(4-cyano- 144 158 (35a) [RD93]3-trifluoromethyl-phenyl)-6-thioxo-5-p-tolyl-5,7-diaza-spiro[3.4]oct-8-ylidene]-thiourea 36-24-[8-(4-hydroxymethyl-phenyl)-5-oxo-7-thioxo-6-aza- 311 337 (36b)spiro[3.4]oct-6-yl]-2-trifluoromethyl-benzonitrile [RD110] 374-[5-(4-formylphenyl)-8-oxo-6-thioxo-5,7- n/a 263 (37a)diazaspiro[3.4]oct-7-yl]-2-trifluoromethyl-benzonitrile [RD114] 384-{5-[4-(1-hydroxyethyl)-phenyl]-8-oxo-6-thioxo-5,7- n/a 187 (38a)diazaspiro[3.4]oct-7-yl}-2-trifluoromethyl-benzonitrile [RD116] 393-{4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6- n/a 197 (39a)thioxo-5,7-diazaspiro[3.4]oct-5-yl]-phenyl}-acrylic [RD117] acid ethylester 40 4-{5-[4-(3-hydroxypropenyl)-phenyl]-8-oxo-6-thioxo- n/a 114(40a) 5,7-diazaspiro[3.4]oct-7-yl}-2- [RD120]trifluoromethylbenzonitrile 41-23-{4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6- No n/a (41b)thioxo-5,7-diazaspiro[3.4]oct-5-yl]-phenyl}-propionic [RD128] acidmethyl ester 41-4 3-{4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6- 224n/a (41d) thioxo-5,7-diaza-spiro[3.4]oct-5-yl]-phenyl}- [RD133]propionamide 41-5 3-{4-[7-(4-Cyano-3-trifluoromethylphenyl)-8-oxo-6- 234n/a (41e) thioxo-5,7-diaza-spiro[3.4]oct-5-yl]-phenyl}-N- [RD134]methyl-propionamide 41-63-{4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6- 732 n/a (41f)thioxo-5,7-diaza-spiro[3.4]oct-5-yl]-phenyl}-N-(2- [RD135]hydroxyethyl)-propionamide 42-24-{4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6- 432 n/a (42b)thioxo-5,7-diazaspiro[3.4]oct-5-yl]-phenyl}-butyric [RD129] acid methylester 42-4 4-{4-[7-(4-Cyano-3-trifluoromethylphenyl)-8-oxo-6- 112 n/a(42d) thioxo-5,7-diaza-spiro[3.4]oct-5-yl]-phenyl}- [RD130] butyramide42-5 4-{4-[7-(4-Cyano-3-trifluoromethylphenyl)-8-oxo-6- 92 n/a (42e)thioxo-5,7-diaza-spiro[3.4]oct-5-yl]phenyl}-N- [RD131] methyl-butyramide43-4 4-[8-Oxo-5-(4-piperazin-1-yl-phenyl)-6-thioxo-5,7- 718 n/a (43e)diazaspiro[3.4]oct-7-yl]-2-trifluoromethylbenzonitrile [RD137] 43-54-{5-[4-(4-methanesulfonylpiperazin-1-yl)-phenyl]-8- 138 n/a (43f)oxo-6-thioxo-5,7-diazaspiro[3.4]oct-7-yl}-2- [RD138]trifluoromethylbenzonitrile 44-2 44-2)3-{4-[7-(4-Cyano-3-trifluoromethyl-phenyl)-8- 113 (44b)oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]-phenyl}- [RD119] acrylamide,(*)No: the compound did not inhibit AR response reporter; (**)n/a: thecompound was not examined in this assay.

One previously unrecognized property of AR overexpression in hormonerefractory prostate cancer is its ability to switch antagonists toagonists. Therefore, only those compounds with minimal or no agonisticactivities are qualified to be anti-androgens for this disease. Todetermine agonistic activities of different compounds, we examined theirstimulating activities on AR using the AR response reporter as themeasure in the LN-AR system in the absence of R1881. Table 2 lists theagonistic activities of different compounds. Consistent with previousresults, bicalutamide activated AR in hormone refractory prostatecancer. The diarylthiohydantoin derivatives such as examples 7-3b(RD37), 33 (RD91), 34 (RD92), and 35 (RD93) have no agonistic activity.In contrast, RU59063, and other anti-androgenic compounds listed asexamples in U.S. Pat. No. 5,705,654, such as examples 30-2, 30-3, 31-2,31-3, and 24-3 (RD73-RD77) strongly activated AR in hormone refractoryprostate cancer.

TABLE 2 Agonistic activities of selective test substances on AR responsereporter in hormone refractory prostate cancer Fold induction byincreasing concentrations of compounds Example Name 0.1 μM 1 μM 10 μMDMSO Dimethyl sulfoxide 1.00(*) 1.00 1.00 R1881 methyltrienolone 44.33n/a(**) n/a Bicalutamide N-[4-cyano-3-(trifluoromethyl)phenyl]-3-[(4-1.06 3.04 10.40 fluorophenyl)sulfonyl]-2-hydroxy-2- methylpropanamide 294-[3-(4-hydroxybutyl)-4,4-dimethyl-5-oxo-2- 10.99 20.84 34.62 Comp.thioxoimidazolidin-1-yl]-2- trifluoromethylbenzonitrile 7-3b4-(8-oxo-6-thioxo-5-(4-methylphenyl)-5,7- 0.87 1.19 0.89 (7c)diazaspiro[3.4]oct-7-yl)-2- [RD37] trifluoromethylbenzonitrile 331-[3-(4-cyano-3-trifluoromethyl-phenyl)-5,5- 1.30 1.18 1.28 (33a)dimethyl-2-thioxo-1-p-tolyl-imidazolidin-4- [RD91]ylidene]-3-ethyl-thiourea 34 1-[7-(4-cyano-3-trifluoromethyl-phenyl)-6-1.19 1.41 1.17 (34a) thioxo-5-p-tolyl-5,7-diaza-spiro[3.4]oct-8- [RD92]ylidene]-3-phenyl-thiourea 351-(4-Cyano-3-trifluoromethyl-phenyl)-3-[7-(4- 1.26 1.10 1.30 (35a)cyano-3-trifluoromethyl-phenyl)-6-thioxo-5-p- [RD93]tolyl-5,7-diaza-spiro[3.4]oct-8-ylidene]-thiourea 30-24-(5-methyl-8-oxo-6-thioxo-5,7- 14.88 19.41 35.22 Comp.diazaspiro[3.4]oct-7-yl)-2 (30b) trifluoromethylenzonitrile [RD73] 30-34-(5-methyl-6,8-dioxo-5,7-diazaspiro[3.4]oct-7- 11.39 14.26 30.63 Comp.yl)-2-trifluoromethylbenzonitrile (30c) [RD74] 31-24-(1-methyl-4-oxo-2-thioxo-1,3- 17.03 16.63 33.77 Comp.diazaspiro[4.4]non-3-yl)-2- (31b) trifluoromethylbenzonitrile [RD76]31-3 4-(1-methyl-2,4-dioxo-1,3-diaza-spiro[4.4]non- 11.99 19.77 38.95Comp. 3-yl)-2-trifluoromethylbenzonitrile (31c) [RD76] 24-34-(4-oxo-2-thioxo-1,3-diazaspiro[4.4]non-3-yl)- 14.88 22.48 37.09 Comp.2-trifluoromethylbenzonitrile (24c) [RD77] (*)Fold induction: activitiesinduced by a specific test substance over activities in DMSO vehicle;(**)n/a: the compound was not examined in this assay.

To examine the specificity of AR inhibitors, selective compounds weretested in LNCaP cells with an over expression of glucocorticoid receptor(GR), the closest member of AR in the nuclear receptor family. Thesecells also carry a GR response reporter and the reporter activity wasinduced by dexamethasone, a GR agonist and the induction was blocked byRU486, a GR inhibitor. Example 7-3b (RD37)(4-(8-oxo-6-thioxo-5-(4-methylphenyl)-5,7-diazaspiro[3.4]oct-7-yl)-2-trifluoromethylbenzonitrile) had no effect on GR in this system.

Effect of Compounds on Ar by Measuring Secreted Levels of ProstateSpecific Antigen (PSA)

It is well established that PSA levels are indicators of AR activitiesin prostate cancer. To examine if the compounds affect AR function in aphysiological environment, we determined secreted levels of endogenousPSA induced by R1881 in the AR-overexpressed LNCaP cells (LNCaP-AR, alsoabbreviated LN-AR). The LNCaP-AR cells are a line of lymph nodecarcinoma of prostate cells transduced with a plasmid that makes expressandrogen receptors. LNCaP-AR cells were maintained in Iscove's mediumcontaining 10% FBS. Two days prior to drug treatment, the cells weregrown in Iscove's medium containing 10% CS-FBS to deprive of androgens.The cells were split and grown in Iscove's medium containing 10% CS-FBSwith appropriate concentrations of R1881 and the test compounds. Afterfour days incubation, secreted PSA levels were assayed using PSA ELISAkits (American Qualex, San Clemente. CA)

The secreted PSA level of LNCaP-AR cells was strongly induced by 25 pMof R1881. In contrast, PSA was not induced in the parental LNCaP cellsuntil concentration of R1881 reached 100 pM. This is consistent with ourprevious report that the AR in hormone refractory prostate cancer ishyper-sensitive to androgens. A dose-dependent inhibition on AR activitywas carried out to determine the IC50s of different compounds ininhibiting PSA expression, and the results were listed in Table 1. IC50sof the selective compounds on PSA expression closely resemble thosemeasured by the reporter assay, confirming that the diarylhydantoinderivatives are strong inhibitors of AR in hormone refractory prostatecancer.

We also examined agonistic activities of selective compounds on AR inhormone refractory prostate cancer using secreted PSA as the surrogatemarker. To do this, androgen-starved AR over expressed LNCaP cells wereincubated with increasing concentrations of the compounds for which asynthesis is described above in the absence of R1881 and secreted PSA inthe culture medium was measured 4 days later.

Table 3 lists the agonistic activities of the selective compounds.Consistent with the results obtained from the reporter assay, thediarylthiohydantoin derivatives such as examples 7-3b (RD37), 33 (RD91),34 (RD92), and 35 (RD93) have no agonistic activities. In contrast,RU59063, and other antiandrogenic compounds listed as examples in U.S.Pat. No. 5,705,654, such as examples 30-2 (RD73), 30-3 (RD74), and 31-2(RD75) stimulated PSA expression in hormone refractory prostate cancer.

TABLE 3 Agonistic activities of selective test substances on endogenousPSA in hormone refractory prostate cancer Fold induction by increasingconcentrations of compounds Example Name 0.1 μM 1 μM 10 μM DMSO Dimethylsulfoxide 1.00(*) 1.00 1.00 R1881 methyltrienolone 20.69 n/a(**) n/aBicalutamide N-[4-cyano-3-(trifluoromethyl)phenyl]-3-[(4- 2.00 2.55 5.55fluorophenyl)sulfonyl]-2-hydroxy-2- methylpropanamide 294-[3-(4-hydroxybutyl)-4,4-dimethyl-5-oxo-2- 6.88 11.50 21.50 Comp.thioxoimidazolidin-1-yl]-2- trifluoromethylbenzonitrile 7-3b4-(8-oxo-6-thioxo-5-(4-methylphenyl)-5,7- 1.25 1.20 1.15 (7c)diazaspiro[3.4]oct-7-yl)-2- [RD37] trifluoromethylbenzonitrile 331-[3-(4-cyano-3-trifluoromethyl-phenyl)-5,5- 1.06 1.30 0.85 (33a)dimethyl-2-thioxo-1-p-tolyl-imidazolidin-4- [RD91]ylidene]-3-ethyl-thiourea 34 1-[7-(4-cyano-3-trifluoromethyl-phenyl)-6-1.31 1.05 0.90 (34a) thioxo-5-p-tolyl-5,7-diaza-spiro[3.4]oct-8- [RD92]ylidene]-3-phenyl-thiourea 351-(4-Cyano-3-trifluoromethyl-phenyl)-3-[7-(4- 1.44 1.30 1.05 (35a)cyano-3-trifluoromethyl-phenyl)-6-thioxo-5-p- [RD93]tolyl-5,7-diaza-spiro[3.4]oct-8-ylidene]-thiourea 30-24-(5-methyl-8-oxo-6-thioxo-5,7- 6.25 17.95 25.65 Comp.diazaspiro[3.4]oct-7-yl)-2- (30b) trifluoromethylenzonitrile [RD73] 30-34-(5-methyl-6,8-dioxo-5,7-diazaspiro[3.4]oct-7- 7.50 15.20 23.75 Comp.yl)-2-trifluoromethylbenzonitrile (30c) [RD74] 31-24-(1-methyl-4-oxo-2-thioxo-1,3- 8.13 18.20 17.50 Comp.diazaspiro[4.4]non-3-yl)-2- (31b) trifluoromethylbenzonitrile [RD75](*)Fold induction: activities induced by a specific test substance overactivities in DMSO vehicle; (**)n/a: the compound was not examined inthis assay.

Effect of Compounds on AR Mitochondrial Activity by MTS Assay

LNCaP-AR cells were maintained in Iscove's medium containing 10% FBS.The compounds were examined for their effect on growth of hormonerefractory prostate cancer cells. Overexpressed LNCaP cells were usedbecause these cells behave as hormone refractory prostate cancer cellsin vitro and in vivo (1). We measured mitochondria activity by MTSassay, a surrogate for growth. LNCaP cells with overexpressed AR (LN-AR)were maintained in Iscove's medium containing 10% FBS. Two days prior todrug treatment, the cells were grown in Iscove's medium containing 10%.CS-FBS to deprive of androgens. The cells were then split and grown inIscove's medium containing 10% CS-FBS with appropriate concentrations ofR1881 and increasing concentrations of the test compounds. After fourdays incubation, cell growth was monitored by MTS (Promega, Madison,Wis.).

Consistent with the reporter assay and PSA assay, growth of theAR-overexpressed LNCaP was stimulated by 25-microM of R1881, but theparental cells were not stimulated until R1881 concentration reached 100microM. FIG. 2 shows the inhibitory effect of selected compounds ongrowth of hormone refractory prostate cancer in the presence of 100 μMof R1881. The current clinical drug bicalutamide did not inhibit hormonerefractory prostate cancer. In contrast, example 5-31) (RD7)(4-[3-(4-methylphenyl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl]-2-trifluoromethyl-benzonitrile) and example 7-3b(RD37)(4-(8-oxo-6-thioxo-5-(4-methylphenyl)-5,7-diazaspiro[3.4]oct-7-yl)-2-trifluoromethylbenzonitrile)inhibited hormone refractory prostate cancer with high potency.

We examined if growth inhibition in the MTS assay occurs by targetingAR, example 5-3b (RD7)(4-[3-(4-methylphenyl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl]-2-trifluoromethyl-benzonitrile)and example 7-3b (RD37)(4-(8-oxo-6-thioxo-5-(4-methylphenyl)-5,7-diazaspiro[3,4]oct-7-yl)-2-trifluoromethylbenzonitrile)were tested in DU-145 cells, a prostate cancer cell line that lacks ARexpression. These compounds had no growth inhibitory effect on DU-145cells. The compounds did not inhibit cells other than AR-expressedprostate cancer cells, as they had no growth effect on MCF7 and SkBr3,two commonly used breast cancer cells, or 3T3, a normal mouse fibroblastcell line.

Examples of in vitro biological activity of diarylthiohydantoinderivatives are shown in the FIGS. 3, 4 and 5. For example, based onrelative luciferase activity, FIG. 3 indicates that at a concentrationof 500 nM the compounds ranked, in order of most active to least activeas follows: RD 152>RD153>RD145>RD163>RD161=RD162>bicalutamide. Forexample, based on relative PSA level, FIG. 4 indicates that at aconcentration of 500 nM the compounds ranked, in order of most active toleast active as follows:RD138>RD131>RD37>RD133>RD134>RD137>RD138>RD135>bicalutamide. Forexample, based on relative MTS units, FIG. 5 indicates that at aconcentration of 500 nM the compounds ranked, in order of most active toleast active as follows: RD168>RD37>RD141>RD162>bicalutamide.

Inhibitory Effect on Hormone Refractory Prostate Cancer Xenograft Tumors

Example 7-3b (RD37)(4-(8-oxo-6-thioxo-5-(4-methylphenyl)-5,7-diazaspiro[3.4]oct-7-yl)-2-trifluoromethylbenzonitrile)was used to examine if the diarylhydantoin derivatives have in vivoeffects on hormone refractory prostate cancer. First we examined thiscompound on xenograft tumors established from AR-overexpressed LNCaPcells. The engineered cells in Matrigel (Collaborative Biomedical) wereinjected subcutaneously into the flanks of the castrated male SCID mice.Tumor size was measured weekly in three dimensions using calipers. Afterxenograft tumors established (tumor size reached at least 40 mm³), micewith tumors were randomized and treated with different doses ofcompounds orally once daily. Consistent with clinical observation,current clinical drug bicalutamide did not inhibit growth of hormonerefractory prostate cancer (same as vehicle) (FIG. 7 a). In contrast,example 7-3b (RD37)(4-(8-oxo-6-thioxo-5-(4-methylphenyl)-5,7-diazaspiro[3.4]oct-7-yl)-2-trifluoromethylbenzonitrile)strongly inhibited growth of these tumors (FIG. 7 a) and the inhibitionis dose-dependent (FIG. 7 b). Furthermore, example 7-3b (RD37) inhibitedPSA expression (FIG. 8), the clinical marker for hormone refractoryprostate cancer.

Example 7-3b (RD37)(4-(8-oxo-6-thioxo-5-(4-methylphenyl)-5,7-diazaspiro[3.4]oct-7-yl)-2-trifluoromethylbenzonitrile)was also tested in another xenograft model of hormone refractoryprostate cancer, hormone refractory LAPC4. This model was establishedfrom passaging of hormone sensitive prostate cancer in castrated mice,which mimics the clinical progression of prostate cancer (2). Similar tothe finding using AR-overexpressed LNCaP xenograft model, currentclinical drug bicalutamide did not inhibit growth and PSA expression inhormone refractory LAPC4 xenograft model (same as vehicle) (FIGS. 9 aand 9 b). In contrast, example 7-3b (RD37) strongly inhibited growth andPSA expression of these tumors (FIGS. 9 a and 9 b).

Inhibitory Effect on Growth of Hormone Sensitive Prostate Cancer Cells

To determine if the diarylthiahydantoin derivatives also inhibit hormonesensitive prostate cancer cells, we tested some selective compounds ongrowth of LNCaP cells by measuring MTS of mitochondria activities. Incontrast to have no effect on growth of hormone refractory prostatecancer, the current clinical drug bicalutamide mildly inhibited hormonesensitive LNCaP cells in a dose-dependent manner. Example 5-3b (RD7)(4-[3-(4-methylphenyl)-4,4-dimethyl-5-oxo-2-thioxoimidazolidin-1-yl]-2-trifluoromethyl-benzonitrile)and example 7-3b (RD37)(4-(8-oxo-6-thioxo-5-(4-methylphenyl)-5,7-diazaspiro[3.4]oct-7-yl)-2-trifluoromethylbenzonitrile)inhibited hormone sensitive prostate cancer with a 10-fold higherpotency than bicalutamide (FIG. 10).

In Vivo Biological Assay

All animal experiments were performed in compliance with the guidelinesof the Animal Research Committee of the University of California at LosAngeles. Animals were bought from Taconic and maintained in a laminarflow tower in a defined flora colony. LNCaP-AR and LNCaP-vector cellswere maintained in RPMI medium supplemented with 10% FBS. 10⁶ cells in100 μA of 1:1 Matrigel to RPMI medium were injected subcutaneously intothe flanks of intact or castrated male SCID mice. Tumor size wasmeasured weekly in three dimensions (length×width×depth) using calipers.Mice were randomized to treatment groups when tumor size reachedapproximately 100 mm³. Drugs were given orally every day at 10 mg/kg and50 mg/kg. To obtain pharmacodynamic readout, the animals were imaged viaan optical CCD camera, 3 hours after last dose of the treatment. A ROIis drawn over the tumor for luciferase activity measurement inphoton/second. The right panels were a representation of the ROIsmeasurements. Data are shown in FIGS. 11 and 12. Over 18 days RD162 waseffective to prevent tumor growth and even to cause tumor shrinkage, andwas distinctly more effective than bicalutamide.

The pharmacokinetics of bicalutamide,4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]-toluene[RD37],N-methyl-4-{4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]phenyl}butanamide[RD131], andN-methyl-4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]-2-fluorobenzamide(52d) [RD162] were evaluated in vivo using 8 week-old FVB mice whichwere purchased from Charles River Laboratories. Mice were divided intogroups of three for each time points. Two mice were not treated withdrug and two other mice were treated with vehicle solution. Each groupwas treated with 10 mg per kilogram of body weight.

The drug was dissolved in a mixture 1:5:14 of DMSO:PEG400:H₂0. (Vehiclesolution) and was administered into mice through the tail vein. Theanimals are warmed under a heat lamp for approximately 20 minutes priorto treatment to dilate their tail vein. Each mouse was placed into amouse restrainer (Fisher Sci. Cat#01-288-32A) and was injected with 200μl of drug in vehicle solution into the dilated tail vein. After drugadministration, the animals were euthanized via CO₂ inhalation atdifferent timepoints: 5 inn, 30 nm, 2 h, 6 h, 16 h. Animals wereimmediately bleed after exposure to CO₂ via cardiac puncture (1 ml BDsyringe+27G ⅝ needle). For oral dosage, the drug was dissolved in amixture 50:10:1:989 of DMSO:Carboxymethylcellulose:Tween80:H20 beforeoral administration via a feeding syringe.

The serum samples were analyzed to determine the drug's concentration bythe HPLC which (Waters 600 pump, Waters 600 controller and Waters 2487detector) was equipped with an Alltima C18 column (3μ, 150 mm×4.6 mm).The RD37, RD131, and RD162 compounds were detected at 254 nm wave lengthand bicalutamide was detected at 270 nm wave length.

The samples for HPLC analysis were prepared according to the followingprocedure:

-   -   Blood cells were separated from serum by centrifugation.    -   To 400 μl of serum were added 80 μl of a 10 μM solution of an        internal standard and 520 μl of acetonitrile. Precipitation        occurred.    -   The mixture was vortexed for 3 minutes and then placed under        ultrasound for 30 minutes.    -   The solid particles were filtered off or were separated by        centrifugation.    -   The filtrate was dried under an argon flow to dryness. The        sample was reconstructed to 80 μl with acetonitrile before        analyzing by HPLC to determine the drug concentration.    -   Standard curve of drug was used to improve accuracy.

The concentration of RD 162 in plasma as a function of time resultingfrom intravenous and from oral administration is shown in FIG. 13. Thesteady state concentration (Css) of bicalutamide, RD131, and RD162 isshown in Table 4. The concentration at steady state of RD162 isessentially as good as that of bicalutamide, and substantially betterthan RD131.

TABLE 4 Steady-state concentration of bicalutamide, RD131, and RD162 inmice plasma. IC50 Css, 10 mg/kg Css, 25 mg/kg Css, 50 mg/kg Name [nM]LogP [μM] [μM] [μM] Bic. 1000 2.91 10.0 11.4 11.9 RD131 92 3.44 0.390.43 0.40 RD162 122 3.20 9.9 10.7 10.2

Ranking of Compounds in Tiers

Tables 5-10 present diarylhydantoin compounds grouped into Tiers 1-6.Table 11 presents diarylhydantoin compounds which have not been placedinto a tier. The placement of compounds into tiers was based onavailable data coupled with analytical judgment. Data consideredincluded in vitro assays (AR response reporter system in LNCaP cellline, PSA level measurement, MTS mitochondrial assay) and in vivoexperiments (tumor size measured directly or by emission induced byluciferase reporter gene, pharmacokinetic assays based on blood plasmalevels). Not every compound was subjected to each assay. Not all datathat was generated is shown. Judgment was applied in ranking compoundsrelative to each other for their utility in treating prostate cancer, inparticular when ranking two compounds for which the same experimentswere not performed. Characteristics considered in establishing theranking include AR antagonism activity, lack of AR agonism in hormonerefractory cells, prevention of tumor growth, tumor shrinkage, andpharmacokinetic behavior, with a longer residence time in blood beingadvantageous.

Tier 1

Generally, Tier 1 compounds are diarylthiohydantoins with adisubstituted left hand aryl ring that are disubstituted on the righthydantoin carbon, and have either an oxygen or N substituent on the lefthydantoin carbon. It is expected that the amido substituent hydrolyzesto an oxygen in aqueous solutions such as encountered in biologicalsystems, in vitro and in vivo. RD100 has good activity with an iodineinstead of a CF₃ substituent on the left hand aryl ring.

Tier 1 compounds (see Table 5) were judged to be much better thanbicalutamide for treating prostate cancer. However, RD37 and RD131 werefound to metabolize fast, that is, have a short residence time in blood.RD 162 had desirable pharmacokinetics.

FIG. 17 shows that under treatment with bicalutamide, PSA levels forLNCaP cells stayed the same or increased relative to treatment withvehicle solution, whereas under treatment with RD162, PSA levelsdecreased. FIG. 18 illustrates that under treatment with vehiclesolution, tumors continued to increase in size. By contrast, undertreatment with RD162 at a dose of 1 mg per kg body weight per day, therate of tumor increase decreased, and the size of the tumor appeared tobe stabilizing after about 17 days. Under treatment with RD162 at a doseof 10 mg per kg body weight per day, tumor size decreased with time.FIG. 19 illustrates that under treatment with RD162 at a dose of 10 mgper kg body weight per day, photon emission associated with luciferaseactivity decreased. FIG. 20 shows that treatment with RD162 at this doseresulted in a decrease or stabilization of tumor size and a decrease inphoton emission associated with luciferase activity.

FIG. 21 shows that under treatment with RD162, RD162′, RD162″, RD169,and RD170 at doses of 100, 200, 500, and 1000 nM, PSA levels of LN-ARcells decreased. Moreover, the higher the dose, the lower the PSA level.FIG. 23 presents urogenital tract weight and rate of photon emissionassociated with luciferase activity initially and after 14 days oftreatment with bicalutamide or with RD162 for intact and castrated mice.The weight and rate of photon emission increased for both intact andcastrated mice. Treatment of castrated mice with RD162 resulted in adecrease in weight and photon emission with respect to the untreatedcastrated mice, as did treatment with bicalutamide.

Thus, Tier 1 compounds are particularly advantageous for use as ARantagonists, and as therapeutic agents for hormone refractory prostatecancer. They may be useful to treat other AR related diseases orconditions such as benign prostate hyperplasia, hair loss, and acne.These and related compounds may also be useful as modulators of othernuclear receptors, such as glucocorticoid receptor, estrogen receptor,and peroxisome proliferator-activated receptor, and as therapeuticagents for diseases in which nuclear receptors play a role, such asbreast cancer, ovarian cancer, diabetes, cardiac diseases, andmetabolism related diseases. They may be useful in assays e.g. asstandards, or as intermediates or prodrugs.

TABLE 5 TIER 1 COMPOUNDS

RD7

RD8

RD10

RD35

RD36

RD37

RD57

RD58

RD90

RD91

RD92

RD93

RD94

RD95

RD96

RD97

RD100

RD102

RD119

RD120

RD130

RD131

RD145

RD152

RD153

RD163

RD162

RD162

RD162

RD168

RD169

RD170

indicates data missing or illegible when filed

Tier 2

Tier 2 compounds (see Table 6) were significantly better thanbicalutamide for treating prostate cancer, although there wereindications that RD54 could act as an agonist. FIG. 3 illustrates thatcompounds RD145, RD152, RD153, RD162, and RD163 in Tier 1 and RD161 inTier 2 dosed at concentrations ranging from 125 nM to 1000 nM acted toreduce luciferase activity in LNCaP-AR cells whereas control solutionsof DMSO and of bicalutamide had little or no effect. FIG. 4 illustrates,for example, that at concentrations of 1000 nM, compounds RD37 andRD131, in Tier 1, caused a greater decrease in PSA level of LNCaP-ARcells than RD133, RD134, and RD138 in Tier 2. FIG. 11 presents tumorvolume over time, and illustrates that under treatment with bicalutamideor vehicle solution, tumors continued to grow, whereas under treatmentwith RD162, in Tier-1, tumors decreased in size. FIG. 12 illustratesthat photon emission associated with luciferase activity remained aboutthe same or increased under treatment with bicalutamide relative totreatment with vehicle solution, whereas photon emission decreased undertreatment with RD162. FIG. 14 illustrates that under treatment withbicalutamide, there was little or no decrease in PSA levels, whereasunder treatment with RD131 and RD162, PSA levels decreased. FIG. 15illustrates that the IC₅₀ for RD37, RD131, and RD162, in Tier 1, wasmuch lower than the IC₅₀ for bicalutamide.

Generally, Tier 2 compounds are structurally similar to Tier 1compounds, but with different substituents on the right hand aryl ring.Tier 2 compounds are advantageous for use as AR antagonists, and astherapeutic agents for hormone refractory prostate cancer. They may beuseful to treat other AR related diseases or conditions such as benignprostate hyperplasia, hair loss, and acne. These and related compoundsmay also be useful as modulators of other nuclear receptors, such asestrogen receptor and peroxisome proliferator-activated receptor, and astherapeutic agents for diseases in which nuclear receptors play a role,such as breast cancer, ovarian cancer, diabetes, cardiac diseases, andmetabolism related diseases. They may be useful in assays e.g. asstandards, or as intermediates or prodrugs.

TABLE 6 TIER 2 COMPOUNDS

RD6

RD13

RD48

RD49

RD51

RD53

RD54

RD55

RD63

RD66

RD68

RD71

RD87

RD103

RD110

RD111

RD114

RD116

RD133

RD134

RD138

RD161

Tier 3

Tier 3 compounds (see Table 7) were judged to be slightly better thanbicalutamide for treating prostate cancer. RD133, RD134, and RD138 (inTier 2) caused a greater decrease in PSA level of LNCaP-AR cells thanRD135 and RD137, in Tier 3. All of these compounds caused a greaterdecrease in PSA level than bicalutamide.

Other Tier 3 compounds (not shown) were not diarylthiohydantoins, andwere comparable in activity to prior art monoarylhydantoin compoundsRD2, RD4, and RD5.

Thus, Tier 3 compounds are useful as AR antagonists, and as therapeuticagents for hormone refractory prostate cancer. They may be useful totreat other AR related diseases or conditions such as benign prostatehyperplasia, hair loss, and acne. These and related compounds may alsobe useful as modulators of other nuclear receptors, such as estrogenreceptor and peroxisome proliferator-activated receptor, and astherapeutic agents for diseases in which nuclear receptors play a role,such as breast cancer, ovarian cancer, diabetes, cardiac diseases, andmetabolism related diseases. They may be useful in assays e.g. asstandards, or as intermediates or prodrugs.

TABLE 7 TIER 3 COMPOUNDS

RD3

RD4

RD5

RD69

RD127

RD128

RD129

RD135

RD137

Tier 4

Tier 4 compounds (see Table 8) were judged to be no better thanbicalutamide for treating prostate cancer. Tier 4 RD 39 and RD40 andTier 1 RD37, for example, differ only in the substituent on the lowerright carbon of the hydantoin ring. The substituents on the right handaryl ring may also affect activity.

Some Tier 4 compounds (including those shown and others that are notshown) were not diaryl compounds (lacking the right hand aryl ring),were not thiohydantoins, were not disubstituted on the carbon on thelower right hand of the hydantoin ring, and/or had substituents otherthan oxygen or amido on the lower left hand carbon of the hydantoinring. This provides evidence of the surprising advantages ofdiarylthiohydantoins that are disubstituted on the lower right handcarbon of the hydantoin ring and have oxygen or amido on the lower lefthand carbon of the hydantoin ring.

Thus, Tier 4 compounds may be useful as AR antagonists, and astherapeutic agents for hormone refractory prostate cancer, at least tothe extent that they are comparable to bicalutamide. They may be usefulto treat other AR related diseases or conditions such as benign prostatehyperplasia, hair loss, and acne. These and related compounds may alsobe useful as modulators of other nuclear receptors, such as estrogenreceptor and peroxisome proliferator-activated receptor, and astherapeutic agents for diseases in which nuclear receptors play a role,such as breast cancer, ovarian cancer, diabetes, cardiac diseases, andmetabolism related diseases. They may be useful in assays e.g. asstandards, or as intermediates or prodrugs.

TABLE 8 TIER 4 COMPOUNDS

RD2

RD9

RD21

RD22

RD23

RD24

RD25

RD26

RD27

RD30

RD31

RD39

RD40

RD44

RD59

RD60

RD67

RD82

RD83

RD117

RD118

RD148

RD149

RD150

RD151

Tier 5

Tier 5 compounds (see Table 9) were inactive or nearly inactive, andthus, were worse than bicalutamide for treating prostate cancer. Thesubstituents on the right hand aryl ring are important to determiningactivity.

Some Tier 5 compounds (some of which are shown and some that are notshown) were not diaryl compounds (lacking the right hand aryl ring),were not thiohydantoins, were not disubstituted on the carbon on thelower right hand of the hydantoin ring, and/or had substituents otherthan oxygen or amido on the lower left hand carbon of the hydantoinring. This provides evidence of the surprising advantages ofdiarylthiohydantoins that are disubstituted on the lower right handcarbon of the hydantoin ring and have oxygen or amido on the lower lefthand carbon of the hydantoin ring. In particular, the terminalsubstituent in RD155, RD156, and 158 (CH₂NR_(x)R_(y), where R_(x,y)=H ormethyl) is not seen as contributing to activity in these compounds.

Tier 5 compounds would not be desirable for treatment of prostate canceror as AR antagonists, although these and related compounds may be usefulas modulators of other nuclear receptors, such as estrogen receptor andperoxisome proliferator-activated receptor, and as therapeutic agentsfor diseases in which nuclear receptors play a role, such as breastcancer, ovarian cancer, diabetes, cardiac diseases, and metabolismrelated diseases. They may be useful in assays e.g. as standards, or asintermediates or prodrugs.

TABLE 9 TIER 5 COMPOUNDS

RD32

RD33

RD65

RD84

RD85

RD155

RD156

RD157

RD158

Tier 6

Tier 6 compounds (see Table 10) were inactive or nearly inactive, andfurthermore were strong agonists, and thus were much worse thanbicalutamide for treating prostate cancer. The comparative compoundsranked very poor relative to the inventive compounds. Notably, RD72 hadvery poor activity, with a chlorine substituent on the left hand arylring, whereas RD7, with a trifluoromethane, and RD100, with iodine,ranked in Tier 1. The results for the Tier 6 compounds provide evidenceof the surprising advantages of diarylthiohydantoins that aredisubstituted on the lower right hand carbon of the hydantoin ring andhave oxygen or amido on the lower left hand carbon of the hydantoinring, and have certain substituents on the left hand aryl ring.

Tier 6 compounds would not be desirable for treatment of prostate canceror as AR antagonists.

TABLE 10 TIER 6 COMPOUNDS

RD72

RD73

RD74

RD75

RD76

RD77

Untiered Compounds

For several compounds, there was insufficient experimental data to rankthem. These untiered compounds are presented in Table 11.

Based on the data and methods of the invention, and applying judgmentbased on review of many compounds, including some not shown here, onecan make some observations about the untiered compounds. Comparativeexample RD1 is expected to be in Tier 3 with comparative examplesRD3-RD5. RD89 is expected to hydrolyze to RD37 (Tier 1), and shouldtherefore have comparable activity. RD104 is expected to hydrolyze toRD58 (Tier 1), and should therefore have comparable activity. RD105 isexpected to hydrolyze to RD8 (Tier 1), and RD139 and RD140 are expectedto hydrolyze to RD138 (Tier 2), and they should therefore havecomparable activity.

TABLE 11 UNTIERED COMPOUNDS

RD1

RD19

RD52

RD79

RD80

RD81

RD89

RD104

RD105

RD106

RD115

RD132

RD136

RD139

RD140

RD141

RD142

RD146

RD147

RD154

In short, novel compounds which show evidence of being far superior tobicalutamide in treating prostate cancer were identified and produced.

Sensitivity of Anti-Cancer Activity of Compounds to StructuralDifferences

The inventors have determined that what might appear to be a smallchange in the structure of hydantoin compounds may result in a largechange in that compound's performance in treating prostate cancer. Forexample, RD161 and RD162 differ only by a single fluorine substituent onan aryl ring, and RD162 is in Tier 1, while RD161 is in Tier 2, bothbeing better than bicalutamide for the treatment of prostate cancer, butRD162 being superior. However, RD149, which differs from RD161 only inhaving an additional carbon atom between the methylcarbamoyl group andthe aryl ring, is no better than bicalutamide for the treatment ofprostate cancer and is ranked in Tier 4. The effect of RD161, RD162, andRD149 on luciferase activity can be seen in FIG. 24. At a givenconcentration of compound, the luciferase activity upon exposure toRD161 and RD162 is less than the luciferase activity upon exposure toRD149.

RD9 differs from RD8 only in that an amino group is substituted for ahydroxyl group. However, whereas RD8 is in Tier 1, much better thanbicalutamide for the treatment of prostate cancer, RD9 is in Tier 4, nobetter than bicalutamide. The effect of RD8 and RD9 on luciferaseactivity in the 1AR cell line can be seen in FIG. 27. For a given dose,the luciferase activity upon exposure to RD8 is less than the luciferaseactivity upon exposure to RD9. The effect of RD8 and RD9 on luciferaseactivity in the 4AR cell line can be seen in FIG. 26. For a given dose,the luciferase activity upon exposure to RD8 is less than the luciferaseactivity upon exposure to RD9. The effect of RD8 and RD9 on PSA levelsin the LN/AR cell line can be seen in FIG. 25. For a given dose, the PSAlevel upon exposure to RD8 is less than the PSA level upon exposure toRD9.

RD130 and RD131 differ from each other only by a methyl substituent onthe end of a carbamoyl group and both compounds are ranked in Tier 1,although RD131 has been found to be particularly advantageous. RD129 isthe same as RD130, with the exception of a methoxy group beingsubstituted for an amino group. However, RD129 is ranked in Tier 3.RD128 is similar to RD129, but has one less carbon in the chain linkingthe ester group to the aryl ring; RD128 is ranked in Tier 3. The effectof RD130, RD131, RD128, and RD129 on PSA levels in the LN/AR cell linecan be seen in FIG. 28. For a given concentration, the PSA level uponexposure to RD130 and RD131 is less than the PSA level upon exposure toRD128 and RD129.

RD153 and RD155 differ from each other in that the former has amethylcarbamoyl group attached to an aryl ring and a dimethylsubstituent attached to the thiohydantoin group, whereas the latter hasa methylamino group attached to the right hand aryl ring and acyclobutyl substituent attached to the thiohydantoin group. WhereasRD153 is in Tier 1, much better than bicalutamide for the treatment ofprostate cancer, RD155 is in Tier 5, inactive or nearly inactive in thetreatment of prostate cancer. The effect of RD153 and RD155 onluciferase activity in the LN/AR cell line can be seen in FIG. 29. For agiven concentration, the luciferase activity upon exposure to RD153 isless than the luciferase activity upon exposure to RD155.

RD58 and RD60 differ from each other in the substitution of a thio foran oxo group and a dimethyl substituent for a cyclobutyl substituent.Whereas RD58 is in Tier 1, RD60 is in Tier 4.

Pharmaceutical Compositions and Administration

The compounds of the invention are useful as pharmaceutical compositionsprepared with a therapeutically effective amount of a compound of theinvention, as defined herein, and a pharmaceutically acceptable carrieror diluent.

The diarylhydantoin compounds of the invention can be formulated aspharmaceutical compositions and administered to a subject in need oftreatment, for example a mammal, such as a human patient, in a varietyof forms adapted to the chosen route of administration, for example,orally, nasally, intraperitoneally, or parenterally, by intravenous,intramuscular, topical or subcutaneous routes, or by injection intotissue.

Thus, diarylhydantoin compounds of the invention may be systemicallyadministered, e.g., orally, in combination with a pharmaceuticallyacceptable vehicle such as an inert diluent or an assimilable ediblecarrier; or by inhalation or insufflation: They may be enclosed in hardor soft shell gelatin capsules, may be compressed into tablets, or maybe incorporated directly with the food of the patient's diet. For oraltherapeutic administration, the diarylhydantoin compounds may becombined with one or more excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like. The diarylhydantoin compounds may becombined with a fine inert powdered carrier and inhaled by the subjector insufflated. Such compositions and preparations should contain atleast 0.1% diarylhydantoin compounds. The percentage of the compositionsand preparations may, of course, be varied and may conveniently bebetween about 2% to about 60% of the weight of a given unit dosage form.The amount of diarylhydantoin compounds in such therapeutically usefulcompositions is such that an effective dosage level will be obtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anyunit dosage form should be pharmaceutically acceptable and substantiallynon-toxic in the amounts employed. In addition, the diarylhydantoincompounds may be incorporated into sustained-release preparations anddevices. For example, the diarylhydantoin compounds may be incorporatedinto time release capsules, time release tablets, and time releasepills.

The diarylhydantoin compounds may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of thediarylhydantoin compounds can be prepared in water, optionally mixedwith a nontoxic surfactant. Dispersions can also be prepared inglycerol, liquid polyethylene glycols, triacetin, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations can contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the diarylhydantoin compounds which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form should be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating thediarylhydantoin compounds in the required amount in the appropriatesolvent with various of the other ingredients enumerated above, asrequired, followed by filter sterilization. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze dryingtechniques, which yield a powder of the active ingredient plus anyadditional desired ingredient present in the previously sterile-filteredsolutions.

For topical administration, the diarylhydantoin compounds may be appliedin pure form. However, it will generally be desirable to administer themto the skin as compositions or formulations, in combination with adermatologically acceptable carrier, which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Other solidcarriers include nontoxic polymeric nanoparticles or microparticles.Useful liquid carriers include water, alcohols or glycols orwater/alcohol/glycol blends, in which the diarylhydantoin compounds canbe dissolved or dispersed at effective levels, optionally with the aidof non-toxic surfactants. Adjuvants such as fragrances and additionalantimicrobial agents can be added to optimize the properties for a givenuse. The resultant liquid compositions can be applied from absorbentpads, used to impregnate bandages and other dressings, or sprayed ontothe affected area using pump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes; gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Examples of useful dermatological compositions which can be used todeliver the diarylhydantoin compounds to the skin are known to the art;for example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S.Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman(U.S. Pat. No. 4,820,508), all of which are hereby incorporated byreference.

Useful dosages of the compounds of formula I can be determined bycomparing their in vitro activity, and in vivo activity in animalmodels. Methods for the extrapolation of effective dosages in mice, andother animals, to humans are known to the art; for example, see U.S.Pat. No. 4,938,949, which is hereby incorporated by reference.

For example, the concentration of the diarylhydantoin compounds in aliquid composition, such as a lotion, can be from about 0.1-25% byweight, or from about 0.5-10% by weight. The concentration in asemi-solid or solid composition such as a gel or a powder can be about0.1-5% by weight, or about 0.5-2.5% by weight.

The amount of the diarylhydantoin compounds required for use intreatment will vary not only with the particular salt selected but alsowith the route of administration, the nature of the condition beingtreated and the age and condition of the patient and will be ultimatelyat the discretion of the attendant physician or clinician.

Effective dosages and routes of administration of agents of theinvention are conventional. The exact amount (effective dose) of theagent will vary from subject to subject, depending on, for example, thespecies, age, weight and general or clinical condition of the subject,the severity or mechanism of any disorder being treated, the particularagent or vehicle used, the method and scheduling of administration, andthe like. A therapeutically effective dose can be determinedempirically, by conventional procedures known to those of skill in theart. See, e.g., The Pharmacological Basis of Therapeutics, Goodman andGilman, eds., Macmillan Publishing Co., New York. For example, aneffective dose can be estimated initially either in cell culture assaysor in suitable animal models. The animal model may also be used todetermine the appropriate concentration ranges and routes ofadministration. Such information can then be used to determine usefuldoses and routes for administration in humans. A therapeutic dose canalso be selected by analogy to dosages for comparable therapeuticagents.

The particular mode of administration and the dosage regimen will beselected by the attending clinician, taking into account the particularsof the case (e.g., the subject, the disease, the disease state involved,and whether the treatment is prophylactic). Treatment may involve dailyor multi-daily doses of compound(s) over a period of a few days tomonths, or even years.

In general, however, a suitable dose will be in the range of from about0.001 to about 100 mg/kg, e.g., from about 0.01 to about 100 mg/kg ofbody weight per day, such as above about 0.1 mg per kilogram, or in arange of from about 1 to about 10 mg per kilogram body weight of therecipient per day. For example, a suitable dose may be about 1 mg/kg, 10mg/kg, or 50 mg/kg of body weight per day.

The diarylhydantoin compounds are conveniently administered in unitdosage form; for example, containing 0.05 to 10000 mg, 0.5 to 10000 mg,5 to 1000 mg, or about 100 mg of active ingredient per unit dosage form.

The diarylhydantoin compounds can be administered to achieve peak plasmaconcentrations of, for example, from about 0.5 to about 75 μM, about 1to 50 μM, about 2 to about 30 μM, or about 5 to about 25 μM. Exemplarydesirable plasma concentrations include at least or no more than 0.25,0.5, 1, 5, 10, 25, 50, 75, 100 or 200 μM. For example, plasma levels maybe from about 1 to 100 micromolar or from about 10 to about 25micromolar. This may be achieved, for example, by the intravenousinjection of a 0.05 to 5% solution of the diarylhydantoin compounds,optionally in saline, or orally administered as a bolus containing about1-100 mg of the diarylhydantoin compounds. Desirable blood levels may bemaintained by continuous infusion to provide about 0.00005-5 mg per kgbody weight per hour, for example at least or no more than 0.00005,0.0005, 0.005, 0.05, 0.5, or 5 mg/kg/hr. Alternatively, such levels canbe obtained by intermittent infusions containing about 0.0002-20 mg perkg body weight, for example, at least or no more than 0.0002, 0.002,0.02, 0.2, 2, 20, or 50 mg of the diarylhydantoin compounds per kg ofbody weight.

The diarylhydantoin compounds may conveniently be presented in a singledose or as divided doses administered at appropriate intervals, forexample, as two, three, four or more sub-doses per day. The sub-doseitself may be further divided, e.g., into a number of discrete looselyspaced administrations; such as multiple inhalations from aninsufflator.

A number of the above-identified compounds exhibit little or noagonistic activities with respect to hormone refractory prostate cancercells. Because these compounds are strong AR inhibitors, they can beused not only in treating prostate cancer, but also in treating other ARrelated diseases or conditions such as benign prostate hyperplasia, hairloss, and acne. Because AR belongs to the family of nuclear receptors,these compounds may serve as scaffolds for drug synthesis targetingother nuclear receptors, such as estrogen receptor and peroxisomeproliferator-activated receptor. Therefore, they may be furtherdeveloped for other diseases such as breast cancer, ovarian cancer,diabetes, cardiac diseases, and metabolism related diseases, in whichnuclear receptors play a role.

The embodiments illustrated and discussed in this specification areintended only to teach those skilled in the art the best way known tothe inventors to make and use the invention. Nothing in thisspecification should be considered as limiting the scope of the presentinvention. All examples presented are representative and non-limiting.The above-described embodiments of the invention may be modified orvaried, without departing from the invention, as appreciated by thoseskilled in the art in light of the above teachings. It is therefore tobe understood that, within the scope of the claims and theirequivalents, the invention may be practiced otherwise than asspecifically described.

1.-22. (canceled)
 23. A method for treating a cancer comprisingadministering a therapeutically effective amount of a compound selectedfrom the group consisting of

or a pharmaceutically acceptable salt thereof to a subject in need ofsuch treatment, thereby treating the cancer.
 24. The method of claim 55,wherein the composition is administered at a dosage of the compound in arange selected from the group consisting of from about 0.001 mg per kgbody weight per day to about 100 mg per kg body weight per day, fromabout 0.01 mg per kg body weight per day to about 100 mg per kg bodyweight per day, and from about 0.1 mg per kg body weight per day toabout 10 mg per kg body weight per day. 25.-26. (canceled)
 27. Themethod of claim 55, wherein the composition is administered at a dosageof the compound of about 1 mg per kg body weight per day. 28.-31.(canceled)
 32. The method of claim 55, wherein the compound isadministered by intravenous injection, by injection into tissue,intraperitoneally, orally, or nasally.
 33. (canceled)
 34. The method ofclaim 55, wherein the compound is in a form selected from the groupconsisting of a solution, dispersion, suspension, powder, capsule,tablet, pill, time release capsule, time release tablet, and timerelease pill. 35.-51. (canceled)
 52. The method of claim 23, wherein thecompound is


53. The method of claim 23, wherein the compound is


54. The method of claim 23, wherein the compound is


55. The method of claim 23, further comprising administering anantiandrogen to the subject.
 56. The method of claim 55, wherein thecompound is


57. The method of claim 55, wherein the antiandrogen is selected fromthe group consisting of a non-steroidal antiandrogen and bicalutamide.58. The method of claim 55, wherein the cancer is selected from thegroup consisting of prostate cancer, hormone sensitive prostate cancer,and hormone refractory prostate cancer.
 59. The method of claim 55,wherein the cancer is prostate cancer and wherein administering thecompound prevents the prostate cancer from progressing to hormonerefractory prostate cancer.
 60. The method of claim 55, wherein thecancer is hormone sensitive prostate cancer and wherein administeringthe compound prevents the hormone sensitive prostate cancer fromprogressing to hormone refractory prostate cancer.
 61. The method ofclaim 55, wherein the cancer is selected from the group consisting ofbreast cancer, hormone sensitive breast cancer, and hormone refractorybreast cancer.
 62. The method of claim 55, wherein the cancer is breastcancer and wherein administering the compound prevents the breast cancerfrom progressing to hormone refractory breast cancer.
 63. The method ofclaim 55, wherein the cancer is hormone sensitive breast cancer andwherein administering the compound prevents the hormone sensitive breastcancer from progressing to hormone refractory breast cancer.
 64. Themethod of claim 55, wherein the cancer is selected from the groupconsisting of ovarian cancer, hormone sensitive ovarian cancer, andhormone refractory ovarian cancer.
 65. The method of claim 23, furthercomprising administering an antiandrogen to the subject, wherein thecompound is administered after administering the antiandrogen.
 66. Themethod of claim 65, wherein the compound is


67. The method of claim 65, wherein the antiandrogen is bicalutamide.68. The method of claim 65, wherein the cancer is selected from thegroup consisting of prostate cancer, hormone sensitive prostate cancer,hormone refractory prostate cancer, breast cancer, and ovarian cancer.69. The method of claim 23, further comprising administering anantiestrogen to the subject.
 70. The method of claim 69, wherein thecompound is


71. The method of claim 69, wherein the cancer is selected from thegroup consisting of breast cancer, hormone sensitive breast cancer, andhormone refractory breast cancer.
 72. The method of claim 69, whereinthe cancer is breast cancer and wherein administering the compoundprevents the breast cancer from progressing to hormone refractory breastcancer.
 73. The method of claim 69, wherein the cancer is hormonesensitive breast cancer and wherein administering the compound preventsthe hormone sensitive breast cancer from progressing to hormonerefractory breast cancer.
 74. The method of claim 69, wherein the canceris selected from the group consisting of ovarian cancer, hormonesensitive ovarian cancer, and hormone refractory ovarian cancer.
 75. Themethod of claim 23, further comprising administering an antiestrogen tothe subject, wherein the compound is administered after administeringthe antiestrogen.
 76. The method of claim 75, wherein the compound is


77. The method of claim 75, wherein the cancer is selected from thegroup consisting of breast cancer and ovarian cancer.
 78. The method ofclaim 23, further comprising administering a receptor modulator selectedfrom the group consisting of an estrogen receptor modulator and anuclear receptor modulator to the subject.